Methods and compounds for raising antibodies and for screening antibody repertoires

ABSTRACT

The present invention relates to compositions and methods for raising antibodies generally comprising 1) providing highly immunogenic vesicles bearing at least one target antigen and 2) immunizing animals with the said antigen-bearing vesicles to induce antigen-specific antibody responses. The invention also relates to methods of screening antibody repertoires comprising 1) providing vesicles bearing at least one target antigen and one marker and 2) isolating antibody-producing cells or particles with defined antigen specificity using the said antigen- and marker-bearing vesicles. Antibodies with defined antigen specificity can then be prepared from isolated antibody-producing cells using known methods of the art. This invention can be used in experimental, research, therapeutic, prophylactic or diagnostic areas.

This application is the US national phase of international applicationPCT/IB2004/000888 filed 13 Feb. 2004 which designated the U.S. andclaims benefit of U.S. Provisional Application No. 60/447,291, filed 14Feb. 2003, the entire contents of each of which are hereby incorporatedby reference.

The present invention relates to compositions and methods for raisingantibodies generally comprising 1) providing highly immunogenic vesiclesbearing at least one target antigen and 2) immunizing animals with thesaid antigen-bearing vesicles to induce antigen-specific antibodyresponses. The invention also relates to methods of screening antibodyrepertoires comprising 1) providing vesicles bearing at least one targetantigen and one marker and 2) isolating antibody-producing cells orparticles with defined antigen specificity using the said antigen- andmarker-bearing vesicles. Antibodies with defined antigen specificity canthen be prepared from isolated antibody-producing cells using knownmethods of the art. This invention can be used in experimental,research, therapeutic, prophylactic or diagnostic areas.

BACKGROUND

Antibodies are cornerstone proteins that play a critical role in thefirst line of defense against environmental intruders includingmicrobes. They are research and diagnosis tools and most importantly canbe used as therapeutic compounds. Numerous companies and researchcenters are today at different stages of development of antibody-basedtherapy candidates and several antibody drugs are already on the market.

The antibodies may be polyclonal or monoclonal. Methods of producingpolyclonal antibodies from various species, including mice, rodents,primates, horses, pigs, rabbits, poultry, etc. may be found, forinstance, in (1). Briefly, the antigen is injected in the presence of anadjuvant (for instance, complete or incomplete adjuvant e.g., Freund'sadjuvant) and administered to an animal, typically by sub-cutaneous,intra-peritoneal, intra-venous or intramuscular injection. Repeatedinjections may be performed. Blood samples are collected andimmunoglobulins or serum are separated.

The first method of producing monoclonal antibodies was established byKohler and Milstein (2). This method is also described in detail, forinstance, in (3). Briefly, this method comprises immunizing an animalwith the antigen in the presence of an adjuvant, followed by therecovery of spleen or lymph node cells, which are then fused withinmortalized cells such as myeloma cells. The resulting hybridomasproduce the monoclonal antibodies and can be selected by limit dilutionsto isolate individual clones.

The preparation of antibodies has become essential for the evaluation ofnovel protein functions and in many cases, antibodies have grown to betherapeutic drugs. The interest for antibodies as therapeutic compoundswas recently revived by the development of technologies for thegeneration of human and humanized antibodies. The many genome-sequencingprograms in progress are providing a wealth of information necessitatingthe systematic preparation of antibodies against novel putative proteindrug targets. This has created a costly bottleneck in the process of newdrug target identification and has emphasized the need for a novelapproach to streamline the process of monoclonal antibody preparation.

Indeed, the basic method of preparing monoclonal antibodies has severallimitations. For instance, the preparation of hybridoma involvestedious, lengthy and inefficient processes of hybrid cells generation,drug-selection, screening and clonal expansion during which rareantibody-producing cells with the desired antigen specificity may belost. Another difficulty is that rapid screening methods have to bedeveloped to identify unique antibody-producing hybridoma in a largepool of hybridomas. These methods may vary with the nature of the targetantigen and its availability. Yet another difficulty is that hybridomascan mainly be generated using cells from mouse or few other non-primateanimals. Therefore, unless mice that are transgenic for the expressionof human antibodies are used, hybridomas yield non-human antibodies thatneed to be transformed into humanized antibodies using recombinant DNAmethods. The later process is required for therapeutic applications andcan only be initiated once clones producing antibodies with desiredspecificity are obtained.

To address these limitations, two types of strategies have emerged,consisting of 1) improving specific steps of the classical approachdescribed above and 2) developing antibody repertoires using recombinantDNA technology. The first strategy yielded, for instance, the SLAMtechnology (4) in which the need for preparation and screening ofhybridoma is eliminated. The second strategy yielded, for instance,phage display technologies in which target proteins react with librariesof bacteriophage expressing antibodies or fragment thereof using abacteriophage panning methods (5). More recently, the HuMY technologywas developed by GeneTastix (U.S. Pat. No. 6,406,863) in which fragmentsof target proteins react with a library of antibody repertoire using atwo-hybrid method in yeast.

Although improvements have been made, each method developed so far stillhas limitations. Notably, a major limitation for most methods is thatthey require large amounts of target protein for animal immunizationand/or screening of hybridoma or bacteriophage libraries. Even in thecase of the SLAM technology, which does not require the preparation andscreening of hybridoma, large amounts of purified target proteins arestill needed to perform plaque assays for isolating antibody-formingcells as described by the authors. The need for purification ofrecombinant proteins in large amounts hampers the rapid validation ofmultiple potential drug target proteins. In many instances, purificationprocess varies from protein to protein, yields insufficient amounts ofmaterial, non-functional or denatured products. The purification processbecomes even more problematic when dealing with membrane proteins orentities consisting of multiple polypeptide complexes.

Some phage display as well as two-hybrid methods use libraries ofantigenic peptides and, thereby, alleviate the need for large-scaleantigen purification. However, one deficiency with these methods is thatantibodies against conformational epitopes are difficult to obtain andfor instance, antibodies restricted to epitopes rising frommulti-polypeptide entities such as anti-MHC/peptide complex antibodiescannot be obtained. Another limitation is that the sizes and qualitiesof the peptide libraries and of the antibody libraries used do notalways allow isolating antibodies with high antigen-specific affinity.This is because affinity maturation of antibodies can only be performedif a first generation antibody is obtained. In contrast, the unlimitedrepertoire of antibody sequences found in mammals combined with thenatural affinity maturation process occurring following repeatedimmunization of these animals provides the most efficient way topotentially generate antibodies against any epitope.

The present invention addresses the limitations of antibody preparationmethods by providing a novel and effective approach. It also providesnew valuable tools to improve existing methods of antibody preparation.

SUMMARY OF THE INVENTION

The present invention now discloses novel methods to produce antibodiesthat combine two technologies related to the display of antigen,adjuvant and/or markers on exosomes and to the use of exosomes as avehicle for inducing potent humoral immune responses and/or forscreening antibody repertoires.

Exosomes are vesicles of endosomal origin that are secreted in theextracellular milieu following fusion of late endosomal multivesicularbodies with the plasma membrane (6,7). Cells from various tissue typeshave been shown to secrete exosomes, such as dendritic cells, Blymphocytes, tumor cells and mast cells, for instance. Exosomes fromdifferent origin exhibit discrete sets of proteins and lipid moieties(8,9). They notably contain proteins involved in antigen presentationand immuno-modulation suggesting that exosomes play a role in cell-cellcommunications leading to the modulation of immune responses. Indeed,exosomes from dendritic cells (DC) pulsed with peptides derived fromtumor antigens elicit anti-tumor responses in animal model using thematching tumor (10,11). Methods of producing, purifying or usingexosomes for therapeutic purposes or as research tools have beendescribed for instance in WO99/03499, WO00/44389 and WO97/05900,incorporated therein by reference. Recombinant exosomes have beendescribed in the art, which derive from cells transfected with plasmidsencoding recombinant proteins. Such recombinant exosomes contain theplasmid-encoded recombinant protein (WO00/28001).

Methods of manipulating the protein content of exosomes and ofdisplaying antigens, adjuvant and markers for therapeutic purposes or asresearch tools have been described in WO03/016522.

The invention relates to a method of isolating single antibody-producingparticles having specificity for a selected antigen, comprising:

1) preparing vesicles, preferably exosomes, displaying a selectedantigen and a marker;

2) contacting or suspending said vesicles of step 1 with an antibodyrepertoire; and,

3) identifying and isolating single antibody-producing particlesreacting with said vesicles.

In a specific embodiment, said antibody-producing particles areantibody-producing cells and said antibody repertoire is a repertoire ofantibody-producing cells. Said antibody-producing cells can be plasmacells, hybridoma or lymphocytes. Preferably, said antibody-producingcells display the produced antibody. Said antibody repertoires areprepared by standard recombinant DNA approaches and that are forinstances found in phage or yeast display libraries. Therefore, in another embodiment, said antibody-producing particles are collection ofphages or yeasts displaying specific antibodies. Said antibody-producingcells can also be antibody-secreting cells.

The invention relates to a method of identifying singleantibody-producing cells having specificity for an antigen comprising:

1) providing antibody-producing cells;

2) preparing vesicles, preferably exosomes, displaying said antigen anda marker;

3) suspending the antibody-producing cells of step 1 with the vesiclesof step 2; and,

4) identifying and isolating single antibody-producing cells reactingwith said vesicles.

Preferably, said antibody-producing cells are lymphocytes. In aparticular embodiment, said lymphocytes are collected from non-humananimals immunized with said antigen.

If antibody-producing cells are antibody-secreting cells, the methodfurther comprises, before the step of suspending the antibody-producingcells with the vesicles, the step of incubating antibody-producing cellswith a first biotinylated-antibody against a ubiquitous cell surfacemarker such as CD81 or CD45, streptavidin and a second biotinylatedantibody directed against immunoglobulin produced by saidantibody-secreting cells. Optionally, said step comprises: 1) incubatingthe antibody-secreting cells with a first biotinylated-antibody againsta ubiquitous cell surface marker and streptavidin; and 2) incubating theresulting antibody-secreting cells with the second biotinylated antibodydirected against immunoglobulin of said antibody-secreting cells.Alternatively, said step comprises: 1) incubating streptavidin with afirst biotinylated-antibody against a ubiquitous cell surface marker anda second biotinylated antibody directed against immunoglobulin of saidantibody-secreting cells; and 2) incubating the resulting streptavidinbearing the first and the second antibodies with the antibody-secretingcells. Preferably, said antibody-secreting cells are selected from thegroup consisting of lymphocytes, hybridoma, and plasma cells.

The present invention discloses methods of isolating singleantibody-producing cells comprising:

1) preparing immunogenic vesicles, preferably exosomes, displaying atleast one antigen or an epitope thereof;

2) raising an antibody response by immunizing a non-human animal withsaid immunogenic vesicles;

3) collecting lymphocytes from an immunized animal;

4) preparing vesicles, preferably exosomes, displaying the said antigenor an epitope thereof of step 1 and a marker;

5) suspending the lymphocytes of step 3 with the vesicles of step 4;and,

6) identifying and isolating single antibody-producing cells reactingwith the vesicles of step 4.

Optionally, said animals have been immunized with immunogens other thanrecombinant exosomes displaying antigens. These immunogens includecommonly used antigen formulation such as purified recombinant antigensin adjuvant, nucleotide-based immunogens (naked-DNA, viral DNA) andcells or cell fractions containing antigens.

The invention relates to a method of isolating particles producing asingle antibody specific of a variant antigen from an antibodyrepertoire comprising:

1) preparing a first population of vesicles, preferably exosomes,displaying said variant antigen and a marker;

2) preparing a second population of vesicles, preferably exosomes,displaying the native antigen and not displaying said marker;

3) suspending said antibody repertoire with the first and secondpopulations of vesicles, the second population being in excess; and,

4) identifying and isolating single antibody-producing particlesreacting with the vesicles of step 1.

The invention relates to a method of identifying cells producing asingle antibody specific of a variant antigen comprising:

1) providing antibody-producing cells;

2) preparing vesicles, preferably exosomes, displaying said variantantigen used for the animal immunization and a marker;

3) preparing vesicles, preferably exosomes, displaying the nativeantigen and not displaying said marker;

4) suspending the antibody-producing cells of step 1 with the vesiclesdisplaying said variant antigen and marker of step 2 and with an excessof the vesicles displaying said native antigen of step 3; and,

5) identifying and isolating single antibody-producing cells reactingwith the vesicles of step 2.

Preferably, said antibody-producing cells are lymphocytes. In aparticular embodiment, said lymphocytes are collected from non-humananimals immunized with said antigen.

The present invention further discloses methods of isolating cellsproducing a single antibody specific of a variant antigen comprising:

1) preparing immunogenic vesicles, preferably exosomes, displaying avariant antigen;

2) raising an antibody response by immunizing a non-human animal withthe said immunogenic vesicles;

3) collecting lymphocytes from immunized animal;

4) preparing vesicles, preferably exosomes, displaying said variantantigen of step 1 and a marker;

5) preparing vesicles, preferably exosomes, displaying the nativeantigen and not displaying said marker;

6) suspending the lymphocytes of step 3 with the vesicles displayingsaid variant antigen and marker of step 4 and with an excess of thevesicles displaying said native antigen of step 5; and,

7) identifying and isolating cells producing a single antibody specificof an antigen variant reacting with the vesicles of step 4.

Optionally, said methods further comprise the following steps: a)recovering DNA or RNA from said selected antibody producing particles,b) amplifying the nucleic acid sequence encoding immunoglobulinsequences or portions thereof, c) cloning the amplified nucleic acidsequence into an expression vector to produce proteins with desiredantigen specificity.

In an embodiment of the above-disclosed methods of isolatingantibody-producing cells according to the present invention, saidantigen displayed by said vesicles is fused to an exosome targetingpolypeptide. In an other embodiment, said antigen displayed by saidvesicles is cross-linked to an exosome targeting polypeptide.

In an alternative embodiment of the above-disclosed methods of isolatingantibody-producing cells according to the present invention, saidantigen is a polypeptide having at least one transmembrane domain andsaid antigen is over-expressed into exosome-producing cells, therebyallowing the generation of recombinant exosomes displaying said antigen.Preferably, said polypeptide having at least one transmembrane domain isa receptor. More preferably, said receptor is a GPCR (G Protein-CoupledReceptor) such as SSTR2, CCR7, CXCR4 and CCR5.

Said antigen can be any protein, for example a receptor or an enzyme, orcompounds other than polypeptides, such as glycolipids, polysaccharides,drugs and organic chemicals. Optionally, said antigen is an orphanreceptor. Optionally, said antigen is a tumor, a viral or a microbialantigen. Alternatively, said antigen can be a MHC complex, moreparticularly a MHC I/peptide complex.

In a preferred embodiment of the above-disclosed methods of isolatingcells producing a single antibody specific of a variant antigenaccording to the present invention, said variant antigen is a mutatedantigen and said native antigen is a wild-type antigen. Optionally, saidvariant antigen is the antigen contacting molecules selected in thegroup consisting of polypeptide, lipid, DNA or small molecule, and saidnative antigen is the free antigen. Preferably said antigen contactingmolecules is MHC/peptide complex. Hence, said variant antigen is anMHC/peptide complex and said native antigen unloaded MHC or MHC loadedwith different peptides. In a particular embodiment, said variantantigen is an HLA-C/HIV peptide complex and said native antigen is anunloaded HLA-C or an HLA-C loaded with different peptides. By differentpeptides is intended peptide different from the first HIV peptide(preferably having less than 50, 30, 20 or 10% identity), for instance apeptide which is not derived from HIV. Alternatively, said variantantigen is ligand-receptor complex and said native antigen is eitherfree ligand or free receptor. In a particular embodiment, said variantantigen includes gp120, CXCR4 and CD4 or gp120, CCR5 and CD4 and saidnative antigen is gp120. Additionally, said variant and native antigenscan be different conformational states of any protein, including anenzyme.

In an other embodiment of the above-disclosed methods of isolatingantibody-producing cells according to the present invention, saidimmunogenic vesicles further display immune accessory molecules.Preferably, said immune accessory molecules are adjuvant polypeptides.More preferably, said adjuvant polypeptides are cytokines such asGM-CSF, IL-2 and CD40L. Said CD40L is preferably a mutated CD40L, saidmutation prevents cleavage and release of soluble CD40L. Optionally,said immune accessory molecules are also fused or cross-linked to anexosome targeting polypeptide. Preferably said soluble immune accessorymolecules such as GM-CSF or IL-2 are fused to an exosome targetingpolypeptide. Alternatively, said immune accessory molecules having atleast one transmembrane domain are incorporated into immunogenicvesicles by over expression into the exosome-producing cells.Optionally, said immune accessory molecules are ligands for specificantigen delivery to antigen-presenting cells.

Optionally, said marker is a detectable molecule such as tags, enzyme,biotin, fluorescent molecules. Optionally, said marker is fused orcross-linked to an exosome targeting polypeptide. Alternatively, saidmarker has a transmembrane domain incorporated into immunogenic vesiclesby over expression into the exosome-producing cells. Additionally, saidmarker is labeled lipids preferentially incorporated in vesicles,preferably exosomes. Preferably said labeled lipids arefluorophore-conjugated lipids such as Rhodamine-DOPE orFluorescein-DOPE.

Optionally, the step of preparing vesicles, preferably exosomes,comprises the following steps:

-   -   a) Providing a genetic construct encoding said antigen;    -   b) Optionally, providing a construct encoding said marker;    -   c) Introducing said construct into exosome-producing cells to        generate recombinant exosomes; and,    -   d) Collecting said recombinant exosomes, wherein said exosomes        carry at their surface antigens encoded by said genetic        construct and, optionally, said marker.

Optionally, several distinct genetic constructs-encoding distinctantigens are introduced into said exosome-producing cells. Preferably,said exosome-producing cells are mammalian cells. More preferably, saidmammalian exosome-producing cells are murine cells.

Alternatively, the step of preparing vesicles, preferably exosomes,comprises the following steps:

a) Providing a molecule comprising said antigen fused to an exosometargeting polypeptide;

b) Optionally, providing a molecule comprising said marker fused to anexosome targeting polypeptide; and,

c) contacting said molecule comprising said antigen, and optionally saidmolecule comprising said marker, with lipid vesicles containingphosphatidyl serine or other lipids naturally contained in exosomes, tocreate functionalized lipid vesicles presenting said antigen, andoptionally said marker, at their surface.

The invention further concerns the isolated antibody-producingparticles, preferably cells, and their use for antibody production, andthe antibodies produced by said antibody-producing particles, preferablycells. The invention also concerns a composition comprising either saidisolated antibody-producing cells or said antibodies produced by saidantibody-producing cells and a pharmaceutically acceptable excipient orcarrier.

The invention further relates to a method of expressing a polypeptidehaving at least one transmembrane domain at the surface of exosomes,comprising:

1) Providing a genetic construct encoding said polypeptide or a portionthereof comprising at least one transmembrane domain;

2) Introducing said construct and over expressing said polypeptide or aportion thereof comprising at least one transmembrane domain intoexosome-producing cells to generate recombinant exosomes; and

3) Collecting said recombinant exosomes, wherein said exosomes carry attheir surface polypeptides encoded by said genetic construct

In a preferred embodiment of this method, said polypeptide having atleast one transmembrane domain is a receptor. More preferably, saidreceptor is a GPCR (G Protein-Coupled Receptor) such as SSTR2, CCR7,CXCR4 and CCR5. Alternatively, said polypeptide is a CD40L, preferably amutated CD40L, said mutation prevents cleavage and release of solubleCD40L.

The invention concerns a functionalized exosome prepared by theabove-described method, a composition comprising said functionalizedexosome and any use of it. More particularly, the invention concerns acomposition comprising said functionalized exosome and apharmaceutically acceptable excipient or carrier. The invention alsoconcerns the use of said functionalized exosome for producing antibodyor for delivering the antigen to a subject.

Therefore, the invention concerns a method of producing an antibody thatbinds a polypeptide having at least one transmembrane domain or anepitope thereof, comprising:

-   -   a) Providing a genetic construct encoding said polypeptide or a        portion thereof comprising at least one transmembrane domain;    -   b) Introducing said construct and over expressing said        polypeptide or a portion thereof comprising at least one        transmembrane domain into exosome-producing cells to generate        recombinant exosomes presenting said polypeptide or epitope at        their surface,    -   c) Collecting said recombinant exosomes and injecting said        exosomes or a portion thereof to a non-human mammal to generate        antibodies that bind said polypeptide or epitope and,    -   d) Collecting antibodies or antibody producing cells from said        mammal.

In a preferred embodiment of this method, said polypeptide having atleast one transmembrane domain is a receptor. More preferably, saidreceptor is a GPCR (G Protein-Coupled Receptor) such as SSTR2, CCR7,CXCR4 and CCR5. Preferably, said exosomes further display a mutatedCD40L, said mutation prevents cleavage and release of soluble CD40L.Antibodies may then be isolated directly from animal serum for thepreparation of polyclonal antibody. Monoclonal antibodies may also beprepared from these animals using traditional approaches includinggeneration of hybridomas or isolation of single antibody-producing cellsby methods such as SLAM.

The invention also concerns a method of delivering an antigen having atleast one transmembrane domain or a portion thereof comprising at leastone transmembrane domain to a subject comprising:

-   -   a) Providing a genetic construct encoding said antigen or a        portion thereof comprising at least one transmembrane domain;    -   b) Introducing said construct and over expressing said antigen        or a portion thereof comprising at least one transmembrane        domain into exosome-producing cells to generate recombinant        exosomes presenting said antigen or said portion thereof at        their surface,    -   c) Collecting said recombinant exosomes and injecting said        exosomes or a portion thereof to said subject.

In a preferred embodiment of this method, said antigen is a receptor.More preferably, said receptor is a GPCR (G Protein-Coupled Receptor)such as SSTR2, CCR7, CXCR4 and CCR5. Preferably, said exosomes furtherdisplay a mutated CD40L, said mutation prevents cleavage and release ofsoluble CD40L.

The invention concerns a method of producing an immune response in asubject against a specific antigen having at least one transmembranedomain or a portion thereof comprising at least one transmembrane domaincomprising:

-   -   a) Providing a genetic construct encoding said antigen or a        portion thereof comprising at least one transmembrane domain;    -   b) Introducing said construct and over expressing said antigen        or a portion thereof comprising at least one transmembrane        domain into exosome-producing cells to generate recombinant        exosomes presenting said antigen or said portion thereof at        their surface,    -   c) Collecting said recombinant exosomes and injecting said        exosomes or a portion thereof to said subject.

In a preferred embodiment of this method, said antigen is a receptor.More preferably, said receptor is a GPCR (G Protein-Coupled Receptor)such as SSTR2, CCR7, CXCR4 and CCR5. Preferably, said exosomes furtherdisplay a mutated CD40L, said mutation prevents cleavage and release ofsoluble CD40L.

The invention further contemplates a method to biosynthetically labelexosomes with fluorophore-conjugated lipids and a method for the cloningof inserts into plasmids without introducing mutation at the cloningsites.

LEGEND TO FIGURES

FIG. 1: ELISA detecting anti-Lactadherin antibody in sera ofLactadherin-immunized mice.

FIG. 2: FACS analysis of recombinant cells transfected withHApC3.1/SSTR2 and labelled with anti-HA tag antibody.

FIG. 3: Western blot analysis detecting HA-SSTR2 expressed in exosomesthat are produced by HApC3.1/SSTR2-transfected cells.

FIG. 4: FACS analysis of a panel of 293 cells transfected with variousGPCR-encoding HApC3.1 and labelled with anti-HA tag antibody.

FIG. 5: Capture-ELISA detecting HA-GPCR expression on exosomes derivedfrom a panel of 293 cells transfected with various GPCR-encodingHApC3.1.

FIG. 6: Capture ELISA detecting HLA-A2 and β2-microglobulin expressionon 293 exosomes.

FIG. 7: Capture-ELISA detecting Reference peptide bound to HLA-A2 onexosomes.

FIG. 8: Western Blot analysis detecting the expression of CD40L andmutCD40L on recombinant exosomes. Western blot analysis revealed thattransfection of 293 cells with pcDNA6-CD40L and pcDNA6-mutCD40L resultedin the expression of CD40L expression in exosomes. Exosomes derived fromCD40L-expressing 293 cells contained the full-length CD40L (FL on lane2) as well as the products of its proteolytic cleavage consisting of thesoluble form of CD40L (SF on lane 2) and the remaining N-terminalextremity that contains the trans-membrane domain of CD40L (TM on lane2). In contrast, exosomes derived from mutCD40L-expressing 293 cellscontained only the full-length form (FL on lane 3). No protein wasdetected in exosomes from parental 293 cells (lane 1).

FIG. 9: DC-maturation assay measuring the biological activity ofrecombinant exosomes displaying CD40L.

FIG. 10: Measurement of fluorescence associated with recombinant exosomeexpressing GFP/C1C2 chimeric protein.

FIG. 11: Capture ELISA detecting fluorescence associated with exosomesderived from cells metabolically labeled with Rh-DOPE.

FIG. 12: FACS analysis detecting PLNC bearing GFP/CCR7-293 exosomes.

FIG. 13: FACS analysis detecting PLNC bearing GFP/A2/MART1-293 exosomes.The percentage of GFP-positive cells in the left PLNC population derivedfrom the side of the mice immunized with A2/MART1-exosomes wassignificantly higher when cells were incubated with GFP/A2/MART1-293exosomes than with GFP/A2-293 exosomes (Panel A, 0.83% positive cells vsPanel B, 0.33% positive cells). In contrast, no significant differencecould be detected when comparing the right PLNC incubated withGFP/A2/MART1-293 and GFP/A2-293 (Panel C, 0.49% positive cells vs PanelD, 0.39% positive cells).

FIG. 14: Gel analysis of single cell-derived RT-PCR products based ontheir ability to bind fluorescent exosomes in an antigen-specificmanner. Two rounds of PCR yielded detectable amounts of PCR productsderived from several single cell cDNA (lane 2, 5, 6, 8 and 13).Sequencing of these PCR products confirmed that they encode the variableregions of immunoglobulins.

FIG. 15: Capture-ELISA detecting recombinant IgG in CHO-culturesupernatants.

FIG. 16: FACS analysis of AbTrap− and + and − hybridoma producinganti-exosome antibodies. Incubation of biotinylated-exosomes withAbTrap− cells resulted in background fluorescence associated with thesecells (panel 1). In contrast, ˜25% of these cells were positive whenbearing the full AbTrap (panel 2).

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses novel methods of raising antibodies andscreening antibody repertoires. The invention more particularly usesmembrane vesicles, of natural or synthetic origin, preferably exosomes,displaying target antigens, co-stimulatory molecules and/or markers.This invention can be used in experimental, research, therapeutic,prophylactic or diagnostic areas.

The present invention stems from the discovery of novel unexpectedproperties of recombinant chimeric proteins containing exosome-targetingdomains and of recombinant membrane receptors. More particularly, theinvention shows that antigens targeted to exosomes are highlyimmunogenic and yield potent humoral immune responses. In addition, itshows that exosomes displaying antigen and a marker can be convenientlyused to isolate antigen-specific antibody-producing cells, even at lowfrequencies of responding cells.

The invention presents many advantages over traditional approaches ofantibody preparation. It is most advantageous when dealing with poorlyimmunogenic antigens, membrane proteins or multi-component polypeptidecomplexes. It is notably suitable when dealing with difficult targetsfor which the preparation of antibody using traditional approach failedor does not produce useful antibodies. Also, the preparation ofantibodies can be achieved without the need to purify large amounts ofantigens. In fact, a single and small-scale purification method toisolate exosomes (U.S. Ser. No. 09/789,748) can be used regardless ofthe nature of the exogenous antigen expressed at their surface. Thus,the antigen preparation step can be completed very rapidly, i.e.,typically within less than 12 hours. This method is rapid and can beperformed on many samples in parallel allowing the simultaneouspreparation of multiple antigens for immunization. The expression ofantigens in a naturally occurring vesicle combined with a gentlepurification procedure helps preserve the native conformation ofantigens, which may enable the generation of relevant antibodies withpotential therapeutic applications. Moreover, the invention generateslipid vesicles that contain a high density of antigen at their surface.This high density can be compared to a polymeric state, which highlyfavours antibody production by increasing antigen avidity. A furtheradvantage of this invention is that the polypeptides can be expressed byexosome producing cells and thus subjected to natural pathways ofprocessing and post-translational modifications (glycosylations, etc.).Another advantage is that antibody-producing cells can be isolated evenwhen low frequencies of responding cells are generated. Such lowfrequencies generally occur following immunization with poorlyimmunogenic antigens or with low amounts of antigens. The sorting of Bcells using exosome-bearing antigens allows isolating very rareantigen-specific antibody-producing cells. An added advantage of thesorting of B cells using exosome-bearing antigens is that the stringencyand specificity of the sorting may be adjusted in order to selectivelyisolate B cells producing antibodies to unique epitopes orconformational epitopes. For instances, subtractive sorting performed inthe presence of an excess of exosome-bearing a receptor and low amountsof traceable exosomes bearing the same receptor bound to itsphysiological ligand may allow isolating B cells producing antibodyreacting with receptor/ligand complex only. Hence, the invention alsoallows generation of smart functional antibodies that can discriminatesubtle conformational changes on the target antigen due to interactionwith other compounds or to mutation/deletion. The latter feature is aconsiderable advantage over other antibody preparation methods.

Immunization with highly immunogenic compounds combined with the highlyselective sorting of unique and rare antibody-producing cells providesan efficient novel method to produce antibodies against definedepitopes.

Method for Raising Antibodies

In this method, antibodies are raised when immunogenic vesicles,preferably exosomes, expressing at least one target antigen areintroduced into an animal. The production of antibody may be evaluatedby testing the serum of immunized-animal by standard approaches. Thepreparation of antibody may be accomplished by affinity purification ofantibodies from the serum for polyclonal antibodies or by isolatingantigen-specific antibody producing cells for monoclonal antibodies. Thelatter may preferably be performed using the method for screeningantibody repertoires described below. Alternatively, monoclonalantibodies may be prepared using the various known approaches ofmonoclonal antibody preparation from immunized animal such as Hybridomascreening and SLAM.

Preparation of Immunogenic Vesicles Displaying Antigens

Immunogenic vesicles displaying antigens are generally prepared byfusing antigen sequences to exosome-targeting domains, preferably theC1C2 domain of Lactadherin. Immunogenic vesicles are natural orsynthetic vesicles such as exosomes and liposomes. Methods for thepreparation of vesicles bearing chimeric proteins that contain exosometargeting domains and their use are described in WO03/016522.

Lactadherin is almost exclusively found associated with exosomes. TheC1/C2 domain of Lactadherin contains a highly specific targeting motiffor exosome surfaces. Therefore, the introduction into a protein of partor the entirety of the C1 and/or C2 domain of Lactadherin or afunctional equivalent thereof allows the targeting of the resultingchimeric protein to exosomes and other lipidic structures.

Therefore, said exosome targeting polypeptide comprises Lactadherin or aportion thereof comprising a functional C1 and/or C2 domain. Inperforming the present invention it is possible to use Lactadherin fromvarious sources or origins. More preferably, said Lactadherin or aportion thereof is a non-human mammalian Lactadherin or a portionthereof. Mammalian Lactadherin includes murine, rat, bovine, porcine andequine Lactadherin, for instance. More particularly, said non-humanmammalian Lactadherin or a portion thereof is selected from: (i) murineLactadherin, (ii) a fragment of murine Lactadherin comprising afunctional C1 and/or C2 domain, and, (iii) a polypeptide comprising atleast 50% primary structure identity with the polypeptides of (i) or(ii). More preferably, said protein is fused to a functional C1/C2domain.

As indicated above, the targeting moiety may be a polypeptide comprisingat least 50% primary structure identity with the polypeptides of (i) or(ii) above. Identity may be determined according to various knowntechniques, such as by computer programs, preferably be the CLUSTALmethod. More preferably, the targeting polypeptide has at least 60%identity, advantageously at least 70% identity with the polypeptides of(i) or (ii). Such Lactadherin variant (or functional equivalent) shouldretain the ability to target polypeptides to exosomes. This property maybe verified as described in the examples, e.g., by creating a chimericgene comprising said variant fused to a marker polypeptide, expressingthe same in an exosome-producing cell and determining the presence ofthe marker polypeptide at the surface of the exosome. PreferredLactadherin variants have at least 85% identity with the polypeptides of(i) or (ii) above. Possible variations include amino acid deletion(s),substitution(s), mutation(s) and/or addition(s).

The amino acid sequence of murine Lactadherin is depicted (SEQ ID No. 1;see also (12), as well as Genbank Accession no. M38337). Optionally,said Lactadherin has an amino acid sequence comprising SEQ ID No. 1 or afragment thereof comprising a functional C1 and/or C2 domain.Preferably, said Lactadherin has an amino acid sequence comprising afunctional C1/C2 domain of SEQ ID No. 1. More preferably, saidLactadherin has an amino acid sequence comprising amino acid residues111-266, 109-266, 271-426, 111426 or 109-426 of SEQ ID No. 1.

Optionally, said exosome targeting polypeptide comprises a functional C1and/or C2 domain of Del-1, Neuropilin-1, coagulation factor 5 orcoagulator factor 8. Optionally, said antigen is fused upstream,downstream or at any internal domain junction of the targetingpolypeptide.

In another particular embodiment, the exosome-producing cells and/or theexosome-targeting polypeptide (preferably the Lactadherin or a portionor variant thereof comprising a functional C1 and/or C2 domain) are fromthe same species as the mammal used for immunization. Indeed, in such asystem, the exosomes and targeting polypeptide are not immunogenic andantibodies are produced essentially only against the selected antigen.

In a particular embodiment, the exosome-producing cells are murinecells, the Lactadherin is a murine Lactadherin or a portion or variantthereof comprising a functional C1 and/or C2 domain, the non-humanmammal is a mouse, and the antigen or epitope is from a differentspecies, for instance of human origin. Even more preferably, the mouseis a humanized mouse, allowing humanized antibodies to be produced.

To that effect, the nucleotide sequence of a protein (the antigen or anepitope) can be fused to the C1 and/or C2 domain of mouse Lactadherinand the resulting chimeric sequence is cloned into a eukaryoticexpression vector using standard molecular biology techniques. Plasmidsencoding the chimeric protein are transfected into an exosome-producingmouse cell line and recombinant exosomes are harvested after severaldays of culture of the transfected cells. Recombinant exosomes are thenpurified by centrifugation on a sucrose gradient (U.S. Ser. No.09/780,748). The presence of chimeric proteins on recombinant exosomesis established by Western blot analysis or ELISA. Recombinant exosomesbearing chimeric proteins are then injected into syngeneic mice togenerate antibodies. In this context, only the antigenic determinantscontained in the protein sequences used to generate chimeric proteinsrepresent foreign antigens in the immunized mice.

Alternative exosome-targeting domains can be screened, identified orselected by the method comprising:

-   -   providing a first genetic construct encoding a candidate        polypeptide, preferably a candidate trans-membrane polypeptide;    -   introducing the first genetic construct into exosome-producing        cells and testing expression of the candidate polypeptide into        exosomes;    -   selecting a candidate polypeptide which is expressed in exosomes        and preparing a second genetic construct encoding said selected        polypeptide fused to a targeted polypeptide;    -   introducing the second genetic construct into exosome-producing        cells and testing expression of the fusion polypeptide into        exosomes; and    -   selecting the polypeptide, which causes efficient expression of        the targeted polypeptide into exosomes.

Our results show that different proteins or polypeptides that containspecific targeting signals directing expression on exosomes can beidentified, selected and/or improved using the above methods. Thesepolypeptides require both the ability to be expressed into exosomes andto target other molecules to such vesicles. These polypeptides may bederived from transmembrane proteins, and may include all or a portion ofsuch proteins, typically a portion comprising at least thetrans-membrane domain. These constructs are particularly suited for thedelivery of antigens to exosomes, particularly receptors andtrans-membrane proteins. Candidate targeting polypeptides may be derivedfrom virtually any protein comprising such a trans-membrane domain, suchas receptors, channels, etc. Specific examples of such targetingpolypeptides include MelanA/MART1, CD40L, CD81, etc., or a portionthereof. The targeting polypeptide may comprise an entire trans-membraneprotein, or only a portion thereof comprising at least onetrans-membrane domain.

Said antigen can be any protein, for example receptors or enzymes, orcompounds other than polypeptides, such as glycolipids, polysaccharides,drugs and organic chemicals. In a first embodiment, said antigens arenon-characterized proteins. Antibodies against said antigens arenecessary in order to identify the function of said proteins and tocharacterize it. Such approach is useful in the post-genomic area.Indeed, all the genomic sequences are known but it is now essential tofind the function of the encoded proteins. Moreover, said antigen can bean orphan receptor. In a second embodiment, said antigen can be rare.Therefore, the preparation of antibodies against said antigen need to bevery efficient. The method according to the present invention isparticularly adapted to such applications.

Other examples of antigens are tumor antigens, viral antigens, andmicrobial antigens, for instance. Illustrative examples of tumorantigens are MAGE, BAGE, Prostate tumor antigens, oncogenes, etc. Theamino acid sequence of these antigens is known per se and can, beproduced by recombinant techniques or by synthesis. Particular antigensto be targeted or presented with this invention include soluble antigensand extracellular domains of receptors. Additional examples of antigensinclude lymphokines (IL-2, IL-4, IL-13), trophic factors (TNF, IFN,GM-CSF, G-CSF, etc.), enzymes, clotting factors, hormones, lipoproteins,etc.

A type of antigens with particular interest is a receptor having atleast one trans-membrane domain, more preferably a GPCR or a portionthereof. Indeed, the invention now allows the preparation of exosomesdisplaying trans-membrane polypeptides with or without any exosometargeting polypeptides. The expression of GPCRs within vesicles allowstheir purification, characterization, the screening for ligands (whethersynthetic or natural), the production of antibodies, etc. A specificexample of a GPCR is, for instance, SSTR2, CCR7, CXCR4 and CCR5 althoughthe invention can be used as well with other receptors.

In another embodiment, natural immunogenic vesicles displaying antigenscontaining at least one trans-membrane domain may be prepared bytransfecting exosome-producing cells and over-expressing the saidtrans-membrane proteins. Indeed, we found that recombinanttrans-membrane proteins may be transferred and expressed in exosomecompartment when they are over-expressed by exosome-producing cells.This unexpected phenomenon was demonstrated for trans-membrane proteinsthat do not occur naturally on exosomes and appears to be restricted tospecific cell lines (as described in Example 2). It is in contrast withthe previous findings demonstrating that protein fusion toexosome-targeting domain is required for expression of soluble proteinsor extracellular domains of receptors on exosomes. Also, in the absenceof exosome-targeting signals, recombinant trans-membrane proteins arefound mainly on the cell surface and only a fraction of the expressedprotein is found on exosomes. The amount found on exosomes appears to bedirectly proportional to the amount of proteins found on the cellsurface.

In yet another embodiment, the epitope profile of proteins expressed onrecombinant exosome may be modified by reacting exosomes with solublecompounds. This can yield multimeric entities consisting for instancesof MHC molecules loaded with a specific peptide or receptor-ligandcomplexes. The objective here is to generate antibodies such asrestricted antibodies that react with MHC/peptide complex but not withunloaded MHC molecules. The method to load MHC molecules with peptide(Direct Loading) was recently described in WO01/82958. Restrictedantibodies are sought after diagnostic and therapeutic agents as theyreact with markers of cancer and infected cells. In addition toclassical MHC molecules, they can be raised against complexes includingother polymorphic entities such as CD1 and non-classical MHC molecules.Restricted antibodies are however very difficult to obtain by classicalantibody preparation methods. An example of application for restrictedantibodies is the generation of compounds capable of killingHIV-infected cells. More specifically, this can be achieved by usingrestricted antibodies that react with HLA-C/HIV peptide complexes.Indeed, HIV has been shown to down-regulate HLA-A and B as one of themany means towards adoptive immune evasion by the virus. However,maintaining HLA-C is also crucial to virus survival as down-regulationHLA-C with A and B subtypes would trigger innate response by NK cells.Hence, it is likely that most HIV-infected cells express HLA-C/HIVpeptide complexes and restricted antibodies against these flags oninfected cells constitute a powerful means to eliminate HIV reservoirsin HIV patients. Therefore, the invention also concerns the use ofrestricted antibodies against HLA-C/HIV for the preparation of apharmaceutical composition for treating and/or preventing HIV infection.The invention further concerns a pharmaceutical composition comprisingsaid antibodies and a method for treating and/or preventing HIVinfection in a subject comprising administering to said subject aneffective amount of restricted antibodies against HLA-C/HIV.

Other examples are to generate antibodies reacting with an active formof a receptor or a ligand (triggered by conformational changes uponreceptor/ligand interaction) but not the inactive form or empty receptoror ligand and reacting with a mutated form of an antigen but not withthe wild type antigen. A typical example here is the preparation ofneutralizing antibodies against HIV. Indeed, neutralizing epitopes havebeen found on HUV gp120 only when this antigen is bound to its receptorson target cells. Hence, generating receptor/ligand complexes includinggp120, CXCR4 and CD4 or gp120, CCR5 and CD4 is required for raisingneutralizing antibodies that inhibit receptor-mediated binding of HIV tocells. In this context, the present invention provides an efficientmeans to prepare appropriate immunogens comprising complex entities thatare maintained in native and functional conformation and generateantibodies against specific conformational epitopes present in thecomplex but not on the separate entities included in it.

The generation and isolation of antibody reacting to subtle epitopes areenabled by further performing contralateral immunization and subtractivesorting of B cells as described below.

In yet another embodiment, immunogenic vesicles displaying antigens mayalso display adjuvant and/or ligand for specific antigen-delivery toantigen-presenting cells. This would provide further mean to increasevesicles immunogenicity, which may result in increasing the strength ofthe humoral responses and thereby the frequency of antigen-specificantibody producing cells. In addition, this may lower even more theamount of antigen required to induce detectable humoral responses.Vesicles with increased immunogenicity may be prepared by transfectingexosome-producing cells with plasmid encoding adjuvant fused toexosome-targeting domains such as GM-CSF/C1C2. Stable cell linesselected for optimum exosome-linked adjuvant activity may be establishedby standard method using drug selection and may be used as recipient fortarget antigen. Methods for the preparation of vesicles with increasedimmunogenicity and bearing chimeric adjuvants that containexosome-targeting domains and their use have been described inWO03/016522.

Alternatively, adjuvant with trans-membrane domain may be displayed onexosomes following over-expression in exosome-producing cells asdescribed above. The class of adjuvant containing a trans-membranedomain includes for instances CD40 ligand (CD40L), a potentimmunostimulator that is produced in a trans-membrane form at thesurface of CD40L-expressing cells and also as a circulating solubleform. We have found that transfection of cell lines with a plasmidencoding CD40L resulted in the expression of this adjuvant on exosomesproduced by transfected cells. Such recombinant exosomes displayedincrease immune potency as described in Example 4. We also found thatthe cleavage site of CD40L yielding the soluble form of this adjuvantmay be mutated to prevent cleavage and release of soluble CD40L fromexosome surface. This results in exosomes expressing the trans-membraneform of CD40L only and that display even more potent immunologicalactivity than exosomes expressing wild type CD40L. A possibleexplanation for this unexpected phenomenon is that the latter contain amixture of full-length and soluble forms of CD40L as well as residualtrans-membrane domain of CD40L, which may hamper the efficaciousformation of functional trimeric form of CD40L. This invention concernsan exosome comprising a CD40L mutated for the cleavage motif.

Therefore, the invention concerns a new method of expressing apolypeptide having at least one transmembrane domain at the surface ofexosomes, comprising:

1) Providing a genetic construct encoding said polypeptide or a portionthereof comprising at least one transmembrane domain;

2) Introducing said construct and overexpressing said polypeptide or aportion thereof comprising at least one transmembrane domain intoexosome-producing cells to generate recombinant exosomes; and

3) Collecting said recombinant exosomes, wherein said exosomes carry attheir surface polypeptides encoded by said genetic construct.

In a preferred embodiment of this method, said polypeptide having atleast one transmembrane domain is a receptor. More preferably, saidreceptor is a GPCR (G Protein-Coupled Receptor) such as SSTR2, CCR7,CXCR4 and CCR5. Alternatively, said polypeptide is a CD40L, preferably amutated CD40L, said mutation prevents cleavage and release of solubleCD40L.

An example of ligand for specific antigen-delivery is immunoglobulin andfragment thereof, such as for instance Fc fragments of immunoglobulins.Such Fc fragments, when expressed at the surface of exosomes, can act totarget the exosomes to cells expressing receptors for such Fc fragments,such as antigen-presenting cells. The expression of such Fc fragments,either alone or in combination with the expression of antigens,facilitates and enhances exosome recognition by antigen-presentingcells, particularly dendritic cells, and increases cross-priming of suchantigens.

Fusions

Chimeric polypeptides or compounds can be prepared by genetic orchemical fusion.

For the genetic fusion, the region of the chimeric gene coding for thepolypeptide of interest may be fused upstream, downstream or at anyinternal domain junction of Lactadherin or a targeting polypeptide.Furthermore, the domains may be directly fused to each other, orseparated by spacer regions that do not alter the properties of thechimeric polypeptide. Such spacer regions include cloning sites,cleavage sites, flexible domains, etc. In addition, the chimeric geneticconstruct may further comprise a leader signal sequence to favorsecretion of the encoded chimeric polypeptide into the endoplasmicreticulum of exosome-producing cells. Moreover, the chimeric gene mayfurther comprise a tag to facilitate purification or monitoring, such asa myc tag, a poly-histidine tag, etc.

For the chemical fusion, the partial or full-length Lactadherin sequencemay be selected or modified to present at its extremity a free reactivegroup such as thiol, amino, carboxyl group to cross-link a solublepolypeptide, a glycolipid or any small molecule. In a preferredembodiment, the Lactadherin construct encodes at least amino acids 1-271of SEQ ID No. 1 in which the C1 domain (amino acids 111-266) providesthe targeting motif to exosomes and Cysteine 271 provides the freethiol-residue for chemical cross-linking to other molecules.Cross-linking peptides, chemicals to SH group can be achieved throughwell-established methods (13). The advantage of this method is that itextends the scope of the invention to the preparation of antibodies tocompounds other than polypeptides, such as glycolipids, drugs andorganic chemicals. It also provides a means to target polypeptide andcompounds to exosomes without introducing putative neo-antigenicdeterminants. Selected cross-linking reagents have been shown to beimmunologically silent (13). Neo-antigenic determinants sometimes occurat the junction of chimeric genes and may limit the usage of chimericgene products for specific prophylactic and therapeutic humanapplications. Modified exosomes or lipid vesicles (e.g., liposomes) canthus be prepared by producing exosomes (or liposomes) presenting therelevant Lactadherin construct such as SEQ ID No. 1 and then reactingthem with the product to be linked. Alternatively, the Lactadherinfragment cross-linked to a product may be prepared and subsequentlyadded to purified exosomes or liposomes.

Vectors

This invention further encompasses a vector comprising a chimericgenetic construct as described above, as well as recombinant cellscomprising a chimeric genetic construct or a vector as described above.The vector may be a plasmid, a phage, a virus, an artificial chromosome,etc. Typical examples include plasmids, such as those derived fromcommercially available plasmids, in particular pUC, pcDNA, pBR, etc.Other preferred vectors are derived from viruses, such as replicationdefective retroviruses, adenoviruses, AAV, baculoviruses or vacciniaviruses. The choice of the vector may be adjusted by the skilled persondepending on the recombinant host cell in which said vector should beused. In this regard, it is preferred to use vectors that can transfector infect mammalian cells. Indeed, preferred recombinant host cells aremammalian cells. These can be primary cells or established cell lines.Mustrative examples include fibroblasts, muscle cells, hepatocytes,immune cells, etc., as well as their progenitor or precursor cells. Mostpreferred mammalian cells are exosome-producing mammalian cells. Theseinclude, for instance, tumor cells, dendritic cells, B and T lymphocytesor mastocytes.

Exosome-Producing Cells

Exosome-producing cells include any cell, preferably of mammalianorigin, that produces and secretes membrane vesicles of endosomal originby fusion of late endosomal multivesicular bodies with the plasmamembrane (6). Cells from various tissue types have been shown to secreteexosomes, such as dendritic cells, B lymphocytes, tumor cells, Tlymphocytes and mast cells, for instance. Methods of producing,purifying or using exosomes for therapeutic purposes or as researchtools have been described for instance in WO99/03499, WO00/44389,WO97/05900, incorporated therein by reference. Preferredexosome-producing cells of this invention are mammalian tumor cells,mammalian B and T lymphocytes and mammalian dendritic cells, typicallyof murine or human origin. In this regard, the cells are preferablyimmortalized dendritic cells (WO94/28113), immature dendritic cells ortumor cells (WO99/03499). Furthermore, for the production of antibody,it may be advantageous to use B lymphocytes as exosome-producing cells,since the resulting exosomes comprise accessory functions and moleculessuch as MHC class II molecules that facilitate antibody production.Furthermore, it has been shown that B cells-derived exosomes are able tobind to follicular dendritic cells, which is another important featurefor antibody induction (14).

The cells may be cultured and maintained in any appropriate medium, suchas RPMI, DMEM, AIM V etc, preferably protein-free media to avoidcontamination of exosomes by media-derived proteins. The cultures may beperformed in any suitable device, such as plates, dishes, tubes, flasks,etc.

The genetic construct (or vector) can be introduced into theexosome-producing cells by any conventional method, such as by naked DNAtechnique, cationinc lipid-mediated transfection, polymer-mediatedtransfection, peptide-mediated transfection, virus-mediated infection,physical or chemical agents or treatments, electroporation, etc. In thisregard, it should be noted that transient transfection is sufficient toexpress the relevant chimeric gene so that it is not necessary to createstable cell lines. The exosomes produced by such cells may be collectedand/or purified according to techniques known in the art, such as bycentrifugation, chromatography, etc. Preferred techniques have beendescribed in WO00/44389 and in U.S. Ser. No. 09/780,748, incorporatedtherein by reference.

Inoculums and Route of Injection

Antibodies are raised following inoculation of antigen to animals,preferably in the form of recombinant exosomes prepared as describedabove. Alternatively, antigens may be administered in a DNA form or in arecombinant protein form. Yet another possibility is to administer wholerecombinant exosome-producing cells.

The antibodies may be polyclonal or monoclonal. Animals can be fromvarious species, including mice, rodents, primates, horses, pigs,rabbits, poultry, etc. Preferred animals are mice.

Genetic immunization can be performed using a variety of viral vectors,such as vaccinia, poxvirus, adenovirus, adeno-associated virus, etc.,non-viral vectors, such as DNA associated with various lipidic orpeptidic compositions, or using pure (e.g., naked) DNA. Various vectordelivery devices or techniques may be used for genetic vaccination,including gene gun or electroporation. As indicated above, the geneticconstruct may be any DNA or RNA molecule, typically a plasmid, viralvector, viral particle, naked DNA or any cell comprising the same. Thevarious genetic constructs may be comprised within a single vector or inseparate vectors or in any combination(s).

Protein immunization may also be used in a similar way. In this respect,recombinant chimeric antigens may be used in a purified form foradministration into the animals.

Following such an administration, chimeric antigens with exosometargeting domain, preferably C1 and/or C2 domain of Lactadherin, will beloaded in vivo on the animal's own circulating exosomes, therebyinducing an immune response.

The inoculum composition generally further comprises a pharmaceuticallyacceptable excipient or vehicle, such as a diluent, buffer, isotonicsolution, etc. The composition may also includetransfection-facilitating agents, as described above for geneticimmunization. The composition can further comprise an adjuvant.

Administration of inoculum can be performed by various routes, such asby systemic injection, e.g., intravenous, intramuscular,intra-peritoneal, intra-tumoral, sub-cutaneous, intra-splenic,intra-nodal etc.

Contralateral immunization may be performed to further increase thelocal frequency of cells producing antibodies reacting to definedepitopes. This approach is very advantageous for the preparation andisolation of antibodies against specific epitopes found for instances onMHC/peptide complex but not on empty MHC molecules or on receptor/ligandcomplex but not on receptor alone. Other examples for this approachconsist of epitopes found on active compounds but not the inactive formof that compound or on a mutated but not the normal form of an antigen.Contralateral immunization was first described as a mean to isolateantibody-producing cells directed against an antigen found in a mixtureof proteins such as cell extracts (15). It is performed by injecting forinstances the one side of an animal with a cell extract alone and thenby later boosting the animal simultaneously with the cell extract aloneat the same site and with the same cell extract containing a targetantigen at the opposite side of the animal. The route of injectiongenerally used for this approach is subcutaneous in the left and rightfootpad of an animal. In this case, antibody-producing cells areisolated from the popliteal lymph node located on the side of the animalthat received the target antigen. In the present invention,contralateral immunization is applied to the preparation and isolationof epitope-specific antibodies by injecting the one side of an animalwith an antigen and the other side with a conformational variant of thatantigen. Conformational variant may result from protein-proteininteractions, mutation/deletion or any modification of an antigen thatresults in the formation of neo-antigenic determinants. In thisinvention, contralateral immunization is best used in combination withsubtractive sorting of B cells described below.

Method for Screening Antibody Repertoires

The invention now discloses a method for the screening of antibodyrepertoires comprising 1) contacting traceable recombinant exosomesbearing a target antigen and a marker with antibody repertoires and 2)isolating antigen-specific antibodies based on their association withthe said marker via exosomes bearing antigens. Antibodies derived fromcell-based library of antibody repertoire and of desired specificity maybe prepared by standard antibody preparation method includingrecombinant DNA and hybridoma. The method is best suited for preparingantibodies from animals immunized according to the method for raisingantibodies described above.

By “antibody repertoire” is intended a population of antibody-producingparticles displaying different antibodies. By “antibody-producingparticles” is intended a particle comprising a nucleic acid encoding anantibody displayed by the particle. Preferably said particle is a cell,a yeast, or a phage. More particularly, said antibody-producing cellscan be plasma cells, hybridoma or lymphocytes. Said antibody-producingcells can also be antibody-secreting cells.

Preparation of Traceable Vesicles Displaying Antigens and a Marker

Traceable vesicles are synthetic or natural vesicles, preferablyexosomes, as described above. They display the target antigen and may bedetected by standard immunological, biochemical or physico-chemicalmethods via for instances, tags, biotin, enzymatic markers orfluorophores.

Methods for the preparation of vesicles displaying antigens have alreadybeen described above. In cases where chimeric proteins were used forimmunization, a second chimeric protein is prepared where the sameprotein antigen sequence is fused with an extended exosome-targetingdomain. Alternatively, the protein antigen may be fused to homologues ofthe exosome-targeting domain derived from a different species. Theobjective here is to create chimeric proteins with new junctionsequences, thereby, avoiding the detection/selection of antibodiesreacting with neo-antigenic determinant that may be found at thejunction of the chimeric protein used for immunization.

Traceable exosomes may be prepared similarly to antigen-displayingexosomes, by transfecting cells with a vector encoding a marker (tags,enzyme, and fluorescent proteins for instances) fused to anexosome-targeting domain or more simply by over-expressing the markerwhen the latter contains a trans-membrane moiety. Exosome displayingboth an antigen and a marker may be prepared using a pre-establishedstable cell line producing traceable exosomes only. The stable cell lineused here may be any laboratory cell line generally used for itscapacity to produce high amounts of recombinant proteins such as 293cells or CHO. This cell line should preferably produce large amounts ofexosomes and should also be derived from a different species than thecell line producing exosome displaying antigens used for immunization.In other terms, when the screening of antibody repertoire is performedusing antibody-producing cells derived from an immunized animal, a mousefor instances, and when antibody responses in this animal were inducedfollowing immunization with mouse-cell line derived recombinant exosome,traceable exosomes displaying the target antigen and a marker used forthe screening of antibody repertoire should be produced by a cell linederived from a species other than mouse. This is to avoidselecting/isolating antibodies that may have been induced againstmouse-derived exosomal proteins and also to avoid the interaction oftraceable exosomes with antibody-expressing cells other than viaantibody-antigen interaction, through ligand-receptor for example.

In another embodiment, traceable exosomes may be prepared by conjugatingactive reagents to exosome such as biotin.

In another embodiment, traceable exosomes may be prepared bybiosynthetic pathways using for instances labeled lipids thatincorporate preferentially in endosomal vesicles including exosomes. Itwas recently shown that incubation of reticulocytes with thefluorophore-conjugated lipid lissamine rhodamine B dioleoyl-phosphatidylethanolamine (Rh-DOPE) resulted in the release of rhodamine-containingvesicles of similar density than exosomes (16). As shown in Example 6,we have found that culture of 293 cells in media supplemented withRh-DOPE or Fluorescein-DOPE yield fluorescent 293-derived exosomes. Incontrast, similar culture of dendritic cells did not yield fluorescentexosomes. This discrepancy may be explained by the different lipidcomposition of exosomes derived from dendritic cells and tumor celllines.

Therefore, the invention concerns a method for preparing traceableexosomes comprising the following steps:

1) incubating exosome-producing cells with fluorophore-conjugatedlipids; and,

2) producing and isolating exosomes from said cells which haveincorporated said fluorophore-conjugated lipids.

Preferably, said fluorophore-conjugated lipids arefluorophore-conjugated DOPE (dioleoyl-phosphatidyl ethanolamine). Morepreferably, said fluorophore-conjugated DOPE is Rh-DOPE orFluorescein-DOPE. Optionally, said exosome-producing cells are 293cells.

Isolation of Antigen-Specific Antibodies

The isolation of antigen-specific monoclonal antibodies is performed byscreening antibody repertoires in which cell-surface antibodiesexpressed by antibody-producing cells are contacted with traceableexosomes bearing the said antigen and a marker prepared as describedabove. Antibody-producing cells are preferably primary or immortalized Blymphocytes. Immortalization may result from transformation of Blymphocytes with transforming agents including viruses and oncogenes orfrom fusion with immortalized cells to prepare hybridoma.Antibody-producing cells may also be libraries of prokaryotic oreukaryotic recombinant cells expressing antibodies, preferably humanantibodies. B lymphocytes may be derived from immunized animals,preferably transgenic animals expressing human immunoglobulins.Immunized animals may be prepared using the present invention forraising antibodies described above or with immunogens other thanrecombinant exosomes displaying antigens. These include commonly usedantigen formulation such as purified recombinant antigens in adjuvant,nucleotide-based immunogens (naked-DNA, viral DNA) and cells or cellfractions containing antigens.

The detection and isolation of individual or population (pools of 2 ormore) of antibody-expressing cells bearing traceable-exosomes may beperformed using techniques known of the art and according to the natureof the antibody-expressing cells and the marker on exosomes used. Forinstances, a rapid and efficient method to isolate single B lymphocyteor a population of B lymphocytes contacting fluorescent exosomes is bysorting cells using a Fluorescent Activated Cell Sorter (FACS). To doso, cells from tissues or organ known to contain B lymphocytes such asblood, spleen and lymph nodes or a fraction thereof are collected fromimmunized animals and incubated with fluorescent recombinant exosomesbearing the antigen used for immunization. An excess of non-fluorescentparental exosomes may also be added to block binding sites other thanantigen-specific sites on B lymphocytes. Fluorescent cells are thenanalysed by FACS and single or pools (2 or more) of fluorescent cellscan be isolated by appropriately adjusting the settings of theapparatus.

In an other embodiment, cells such as plasma cells or hybridoma that donot express antibodies at their cell surface but only produce solubleantibodies that are released in the extracellular milieu may be isolatedusing traceable-exosomes as well. In this case, an antibody trap isformed at the cell surface to capture secreted antibodies as they arereleased, before incubating antibody-expressing cells withtraceable-exosomes. Antibody traps may comprise for instances, a firstbiotinylated-antibody against a ubiquitous cell surface marker such asCD81 or CD45, Streptavidin and a second biotinylated antibody directedagainst immunoglobulin specific of the lymphocytes species. For example,if the antibody-producing cells are from mouse, the second biotinylatedantibody is directed against mouse immunoglobulin. The trap is preparedby successfully incubating cells with the first biotinylated anti-cellsurface marker antibody and Streptavidin. The remaining freebiotin-binding sites on Streptavidin can then be used to bridge thesurface of antibody-producing cell and an antibody-capturing moiety,i.e. the second biotinylated anti-immunoglobulin antibody.Alternatively, antibody traps can be prepared using preformed bridgecontaining Streptavidin bearing both biotinylated antibodies. Antibodytrapping occurs during culture of cells to allow release of endogenouslyproduced antibody and its capture at the cell surface of producingcells. The detection and isolation of individual or population (pools of2 or more) of antibody-secreting cells may be performed usingtraceable-exosomes as described above. Of course, this method can alsobe performed with means equivalent to the couple biotin-streptavidin.

The invention also relates to the isolation of soluble antibodies thatmay be derived from blood of immunized animals or a fraction thereof.Soluble antibodies may also be derived from any expression systemproducing recombinant antibodies individually or as pools or librariesof recombinant antibodies with various antigen specificities. Here,recombinant exosomes expressing antigens are used as vehicle to isolateantibodies by a classical method of affinity-purification.

In another embodiment, the invention also relates to the isolation ofepitope specific or conformation-specific antibodies. Such antibodiesmay react specifically to a variant form of a given antigen but not tothe native form of that antigen. Variant and native antigens may berepresented by mutated and wild type antigen or by antigen contactinganother polypeptide, a lipid, DNA or a small molecule and free antigen.Examples of the latter are MHC/peptide complexes and unloaded MHC or MHCloaded with different peptides. A preferred MHC/peptide complexaccording to the present invention is the HLA-C/HIV peptide. Anotherclass of such variants and native antigens is ligand-receptor complexand free ligand or free receptor. Preferred ligand receptor complexesaccording to the present invention are a gp120, CCR5, CD4 complex and agp120, CXCR4, CD4 complex. Antigen variants may also result fromphysiological processes yielding non-constitutive forms of a givenantigen. These include transient conformational states of antigensinduced by activation/deactivation pathways involving modifying enzymessuch as kinases, proteases, glycosylases and phosphatases. Isolation ofepitope-specific or conformation specific antibodies may be performed bysubtractive screening where a variant antigen bearing the targetedepitope is expressed on traceable exosomes and the native form of thesaid antigen lacking the targeted epitope is expressed on non-traceableexosomes. When added in excess to antibody-producing cells, the latterallows the blocking of binding sites on cells producing antibodies thatreact to common epitopes on the variant and native form of the saidantigen. Hence, when traceable exosomes expressing variant antigen andan excess of non-traceable exosomes expressing native antigen are addedsimultaneously, cells producing antibodies directed to specific epitopesfound on the variant antigen react more likely to the traceableexosomes. Thereby, epitope-specific or conformation-specific may beselectively isolated. Subtractive isolation of epitope-specific orconformation-specific antibodies is better used when antibody-producingcells are derived from immunized-animals receiving contra lateralinjection as described above since the cell population collected fromthese animals is also enriched in epitope-specific orconformation-specific antibody-producing cells.

Production of Antigen-Specific Antibodies

The nucleotide sequences encoding at least the variable regions of heavyand light chains of immunoglobulins produced by the antibody-producingcells isolated as described above may then be cloned into expressionvectors for their production as full-length or fragments of recombinantantibodies. To do this, RNA is extracted from single or pools (2 ormore) of selected antibody-producing cells and nucleotide sequencesencoding antibodies are amplified using standard RT-PCR methods withappropriate primers. The PCR products are then cloned into expressionvectors. When only the variable regions of immunoglobulins areamplified, the cloning may be performed in a vector recipient alreadycontaining the full-length or part of the matching leader sequence andconstant regions of heavy and light chains of immunoglobulin, preferablyof human origin Production of recombinant antibodies or fragmentsthereof is then performed by transfecting cells with a mixture of DNAplasmids encoding the matching heavy and light chain of immunoglobulins.Alternatively, both chains may be cloned into a unique plasmid allowingthe simultaneous expression of two gene transcripts. When heavy andlight chain-PCR products are derived from more than oneantibody-producing cell, a matrix of different combinations of heavy andlight chain DNA plasmids may be created to reconstitute immunoglobulinswith the appropriate antigen specificity. The recipient cells forexpression of recombinant antibodies may be any cell line capable ofproducing recombinant proteins such as CHO, 293 or myeloid cell lines.Expression of recombinant immunoglobulins may be determined by standardmethods such as ELISA. Antigen-specificity of recombinantimmunoglobulins may also be verified by standard immunological methodsaccording to the nature and availability of the target antigen. Forinstances, ELISA may be performed using recombinant exosomes expressingantigen as described above. Antibody sequences may be furthermanipulated by recombinant DNA techniques to increase the affinity ofthe antibody for its target antigen, a process known as antibodymaturation, or to humanize the antibody for therapeutic applications.

In another embodiment, the invention relates to the usage of recipientvectors for the cloning of variable sequences of immunoglobulins thatcontain a cassette framed by two restriction sites recognized byrestriction enzymes type IIs, such as BsmB I. These type IIs enzymes cutdouble stranded DNA outside their recognition sites, which allowsinsertion of DNA fragments using overlapping bases of the insertsequence, while conventional cloning uses overlapping bases ofrestriction sites usually added at the extremities of the insert. Hence,the invention allows cloning of variable sequences between the leadersequence and constant region of immunoglobulins without introducingmutations at the cloning sites. In contrast, most conventional cloningmethods of variable sequences introduce mutation that may modify theaffinity of the recombinant immunoglobulin. Example 10 further detailsthe recipient vector and the advantages of this invention.

Therefore, the invention concerns a method of cloning of a variablesequence between the leader sequence and constant region ofimmunoglobulins without introducing mutations at the cloning sitescomprising the steps of:

1) providing a vector containing sequentially said leader sequence, acloning cassette framed by two restriction sites recognized byrestriction enzymes type II such that the restriction sites are removedfrom said vector after restriction enzyme cleavage, and said constantregion;2) providing said variable sequence;3) incubating said vector with said restriction enzyme;4) ligating said variable sequence and said vector resulting from step3.5) isolating said vector comprising said variable sequence between saidleader sequence and said constant sequence.

In another embodiment, isolated antibody-producing cells may be expandedin vitro by culturing them in appropriate medium. Alternatively, thesecells may be immortalized by transformation or fusion as describedabove. Antibodies produced thereby are then purified from culturesupernatants by methods known to the art such as chromatography usingProtein-G beads.

In yet another embodiment, polyclonal antibodies may be purified fromserum by affinity purification using for instances recombinant exosomescoupled to a solid support. Antibody bound to the solid support throughantigen-specific interaction may be eluted using standard method ofantibody elution, i.e. low pH.

In a more general aspect, the invention concerns a method of cloning ofa sequence in a vector without introducing mutations at the cloningsites comprising the steps of:

1) providing a vector containing a cloning cassette framed by tworestriction sites recognized by Type IIs restriction enzymes such thatthe restriction sites are removed from said vector after restrictionenzyme cleavage;

2) providing said sequence with compatible extremities;

3) incubating said vector with said restriction enzyme;

4) ligating said sequence and said vector resulting from step 3.

5) isolating said vector comprising said sequence.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Exosomes are Potent Novel Vehicle for Raising Antibodies

Nine Balb/C mice were arranged in three immunization groups of threemice. Each mouse was immunized intraperitoneally with either ˜20 ngrecombinant human Lactadherin in PBS (Group 1), ˜20 ng recombinant humanLactadherin in a 1:1 PBS/Complete Freund's Adjuvant mix (Group 2) orrecombinant WEHI exosomes containing ˜20 ng human Lactadherin in PBS(Group 3).

Human recombinant Lactadherin was prepared from recombinant exosomesproduced by CHO cells transfected with the plasmid pcDNA6hLactlf/Hisencoding the full-length recombinant human Lactadherin fused to a (His)₆tag (SEQ ID No. 2). pcDNA6hLactlf/His was prepared as follows. Twooverlapping fragments of human Lactadherin cDNA were amplified fromhematopoietic cell-derived cDNA using primer pairs LTDNf15(SEQ ID No.3)/LTDNr8(SEQ ID No. 4) and LTDNf2(SEQ ID No. 5)/LTDNr13(SEQ ID No. 6),respectively. LTDN/f15 and LTDNr13 were extended at their 5′ end toinclude a Hind III and an Age I restriction site whereas LTDNr8 andLTDNf2 included an EcoR I site. The amplification of the 3′ end fragmentof Lactadherin cDNA with LTDNf2/LTDNr13 yielded multiple products, thelongest of which corresponding to the Lactadherin cDNA. The 5′ endfragment was digested with Hind III and EcoR I whereas the 3′ endfragment was digested with Age I and EcoR I. The 5′ end and 3′ endfragments were ligated together and into pcDNA6A-His (Invitrogen) thatwas precut with Hind III and Age I to yield pcDNA6hLactlf/His. Thisplasmid was transfected into CHO cells, a hamster ovarian cell line(ATCC) using lipofectamine (Invitrogen). At day 1 of culture in completemedia (CHO-SFM supplemented with 2 mM L-glutamine, 100 U/ml Penicillin,0.1 mg/ml Streptomycin and 2% fetal bovine serum (FBS)) at 37° C. in a5% CO₂ atmosphere, stably transfected cells were selected in mediasupplemented with 2 μg/ml Blasticidin. After 4 days of culture, stableclones were isolated by the limiting dilution technique. Clonesproducing large amounts of Lactadherin were selected by Western blotanalysis of recombinant Lactadherin expressed in exosomes as follows.Culture supernatants were harvested and successively spun at 200 g andfiltered through a 0.2 μm filter to remove cell debris. The clearedsupernatants were then spun at 4° C. for 90 min. under 100,000 g topellet exosomes. The pellets were resuspended into 100 μl of ice-coldPBS. Eight μl of SDS-PAGE Sample Buffer 5× (SB) was added to thirty-twoμl of exosome in PBS, incubated at 10° C. for 5 min. then analyzed bySDS-PAGE. Proteins on the gel were transferred to PVDF membranesfollowing semi-dry electro-transfer. The presence of human Lactadherinin the samples was established by immunodetection using a 1/2500dilution of polyclonal antibody directed to the RGD motif of humanLactadherin (a gift from Dr. Sebastian Amigorena). Antibody bound toLactadherin was detected using a 1/5000 dilution of secondaryanti-rabbit IgG antibody conjugated to horse-radish peroxidase (JacksonImmunoResearch) and a colorimetric substrate (CN/DAB, Pierce). The cloneCHO-3.2 was selected for production and expanded into 1-liter spinnerflask in complete media without FBS for large-scale production ofLactadherin. Seven-day cell culture supernatant was transferred into250-ml centrifuge bottles and spun 5 min at 2000 rpm to pellet cells.The supernatant was then filtered through 0.2 μm filter and concentratedto 100 ml using a fiber cartridge with a 500 KD size cut-off.Concentrated supernatant was then spun under 100,000 g for 1 hour 15 minat 4° C. The pellet containing exosome was resuspended in 1 ml MLBII (50mM NaPO4 pH 8/300 mM NaCl/10 mM imidazole/0.5% Tween) and transferredinto a tube containing 2 ml Ni-NTA slurry (prespun to remove EtOH).After an incubation of 2-3 hours at 4° C. on a shaker, the sample waspoured into a BioRad column and allowed to settle at 4° C. The columnwas washed with 10 ml MWBI (50 mM NaPO4 pH 8/300 mM NaCl/20 mMimidazole/0.05% Tween then with 20 ml MWBII (50 mM NaPO4 pH 8/500 mMNaCl/20 mM imidazole). Proteins bound to the column were eluted with 8ml MEBII (50 mM NaPO4 pH 8/300 mM NaCl/250 mM imidazole). Elutedproteins were concentrated and buffer was exchanged to PBS pH 7.4 usinga Millipore Ultrafree-4 10,000 MWCO device. The protein sample wasaliquoted and stored at −20° C. This procedure yields highly purifiedrecombinant Lactadherin.

Exosomes derived from the mouse cell line WEHI and expressingrecombinant human Lactadherin were prepared exactly as described aboveusing CHO cell lines except that exosomes were collected after 4 days ofculture of transiently transfected cells. Samples for injections inimmunization group 1 to 3 were normalized for the amount of recombinantLactadherin injected per mouse. Normalization was established by Westernblot analysis as described above using serial dilutions of recombinantLactadherin and recombinant exosomes.

Animals received one boost two weeks after the first injection with thesame samples except group 2 where the antigen was resuspended in a 1:1PBS/Incomplete Freund's Adjuvant mix. Animals were bled after the secondimmunization and tested for anti-human Lactadherin antibody by ELISA.For the ELISA, 50 ng human Lactadherin in PBS was coated to the wells ofa microtitration plate for one hour at 37° C. Blocking buffer containing0.05% Tween-20 and 6% Non-Fat Dry Milk in PBS was added to the wells forone hour at room temperature (RT) to saturate the remaining free bindingsites. Wells were then incubated for one hour at RT with serum ofimmunized mice at a dilution 1/1000 in Blocking buffer. After washingthe wells three times with Blocking buffer, bound antibodies weredetected using a 1/10000 dilution of secondary anti-mouse IgG conjugatedto horse-radish peroxidase (Jackson ImmunoResearch) and a ECL substrate(Amersham). The results of this ELISA are shown in FIG. 1.

Results: Anti-Lactadherin antibodies were detected in the serum of miceimmunized with human Lactadherin-coated mouse exosomes whereas noantibody response was generated when human Lactadherin was given aloneor as an emulsion in Freund's Adjuvant. No antibody was detected whenusing Freund's adjuvant even after four injections of the inoculumwhereas the titer of antibody in serum of mice receivingLactadherin-bearing exosomes increased with subsequent injections (datanot shown).

Conclusion: Exosomes bearing antigens are highly immunogenic in theabsence of any adjuvant and can induce an antibody response using verylow amounts of antigens, amounts at which a classical and already potentadjuvant such as Freund's Adjuvant is inefficient. Hence, recombinantexosomes displaying antigens are powerful tools to raise antibodiesagainst said antigens.

EXAMPLE 2 Method for the Generation of Exosomes Expressing RecombinantMembrane Proteins

The cDNA encoding the Somatostatin receptor (SSTR2), a G Protein-CoupledReceptor (GPCR), and derived from human brain RNA (Clontech, CA) wasamplified by PCR using the primers SSTR2f1 (SEQ ID No. 7) andSSTR2r2(SEQ ID No. 8). The primers contained 5′ end extensions with aHind m restriction site for SSTR2f1 and a Not I restriction site forSSTR2r2. Following digestion with Hind m and Not I, the PCR product wascloned into HApC3.1, a modified pcDNA3.1 (Invitrogen, CA) to prepare theexpression vector HApC3.1/SSTR2 (SEQ ID No. 9). HApC3.1 contains a HAtag-insert (SEQ ID No. 11) that was obtained synthetically (Genset, CA)and cloned into pcDNA3.1 between the Nhe I and Hind III sites of thevector. The HA tag encodes an initiation codon followed by a stretch ofcodons derived from the hemagglutinin antigen sequence and HApC3.1/SSTR2encodes HA-SSTR2, a recombinant SSTR2 with the HA tag at its N-terminalextremity (SEQ ID No. 10). HApC3.1/SSTR2 was transfected into the mousecell lines EL-4 and B3-Z by electroporation (under 220v, 950 microF withBioRad GenePulser electroporator). Transfected cells were cultured inselection media (RPMI 1640-2 mM Lglutamine, 100 U/ml Penicillin, 0.1mg/ml Streptomycin, 1 mM Sodium Pyruvate and 10% fetal bovine serum(FBS)) supplemented with the antibiotic G418 (Invitrogen) at 1 mg/ml forover 7 days at 37° C. in a 5% CO₂ atmosphere. Cells were harvested forFACS analysis whereas culture supernatants were collected for exosomepreparation. For FACS analysis, 10E6 transfected and parental cells wereincubated for 1 hour at 4° C. in 1 ml PBS/5% FBS with anti-HA antibodyconjugated to FITC (Roche, CA). After several washes in PBS/5% FBS cellswere analyzed by FACS to evaluate the expression of HA-SSTR2 at theirsurface. The profile of HA-SSTR2 expression on ELA and B3-Z cells isshown FIGS. 2A and B, respectively.

Exosomes were prepared from culture supernatants as described inExample 1. Exosome production and the presence of HA-SSTR2 weredetermined by Western Blot analysis also as described in Example 1 usingan anti-Actin (Sigma, MO) and an anti-HA antibody (HRP-conjugate, Roche,Calif.) as detecting antibodies, respectively. Exosomes from recombinantEL-4 and B3-Z were analyzed in lane 1 and 2 of FIG. 3. Detectingantibodies were anti-Actin antibody in panel A and anti-HA antibody inpanel B of FIG. 3.

A panel of cDNA encoding other GPCR including CXCR4, CCR5 and CCR7 wasalso cloned into HApcDNA3.1 using the same cloning strategy describedabove for SSTR2. The primer pairs CXCR4f1 (SEQ ID No. 10)/CXCR4r2(SEQ IDNo. 11), CCR5f1 (SEQ ID No. 12)/CCR5r2(SEQ ID No. 13) and CCR7f7(SEQ IDNo. 14)/CCR7r8(SEQ ID No. 15) were used to produce PCR products encodingCXCR4, CCR5 and CCR7, respectively, using cDNA derived from humanhematopoietic cell culture. The resulting plasmids HApcDNA3.1/CXCR4 (SEQID No. 16), HApcDNA3.1/CCR5 (SEQ ID No. 20) and HApcDNA3.1/CCR7 (SEQ IDNo. 22) encode the recombinant protein HA-CXCR4 (SEQ ID No. 19), HA-CCR5(SEQ ID No. 21) and HA-CCR7 (SEQ ID No. 22), respectively. Theseplasmids as well as HApcDNA3.1/SSTR2 were transfected into the humanembryonic cell line 293 cells by electroporation (220V, 950 μF on aBioRad GenePulser). Stable transfectants were established in cultureswith 293-SFM media supplemented with 4 mM L-glutamine, 100 U/mlPenicillin, 0.1 mg/ml Streptomycin, 2% FBS and 250 μg/ml G418.Expression of recombinant protein containing HA tags at the cell surfaceand on exosomes of transfectants was monitored by FACS analysis asdescribed above and capture ELISA, respectively. The profile of HA-GPCRexpression on transfected 293 cells is shown FIG. 4. For capture ELISA,the wells of a microtitration plate were coated with 100 ng of anti-CD81capture antibody (PharMingen) for 1 hour at 37° C. The plate was washed3 times with 200 μL/well of PBS/0.05% Tween20 then incubated at roomtemperature (RT) for 1 hour with 200 μL/well of DPBS/1% BSA. Following awashing step, purified exosomes prepared as described in Example 1 wereadded to the wells. After an incubation overnight at RT with shaking,the wells were washed 3 times with washing buffer. Detecting antibodywas then added to the wells and measured by subsequently adding eithersecondary antibody-HRP conjugates or Streptavidin-HRP when a detectingantibody conjugated to biotin is used, followed by chemiluminescentsubstrate (Amersham). A normalization assay was performed to quantifyexosomes based on the measurement of CD81 using serial dilution of theexosome preparations and a detecting anti-CD81 antibody conjugated toHRP. A second assay was then performed to evaluate expression ofrecombinant antigens using equal and unsaturating amounts of exosomesand anti-HA antibody. The results of the second assay are shown FIG. 5.

Results: FIG. 2 reveals that anti-HA antibody detected specificallyrecombinant HA tag-containing proteins at the surface of both EL4 andB3Z transfected with HApcDNA3.1/SSTR2 whereas this antibody did not bindto the parental EL4 and B3Z cells. HA-tag containing protein could alsobe detected by Western blot in exosomes derived from stably transfectedEL-4 cells (FIG. 3A, lane 1) supporting that recombinant SSTR2 can beexpressed on exosomes without requiring fusion to exosome-targetingdomains. In contrast, the same analysis did not allow the detection ofHA-SSTR2 in exosomes from stably transfected B3Z cells (FIG. 3A, lane2). Exosome production by both cell lines was verified by Western blotanalysis using an antibody against Actin, a constitutive component ofexosomes (FIG. 3B, lane 1 and 2). Similar results were obtained whenusing an anti-SSTR2 antibody to detect HA-SSTR2 expression on cells andexosomes (data not shown).

Transfection of 293 cells with HA-pcDNA3.1/GPCR also resulted inexpression of HA tag-containing protein on the cell surface oftransfected cells. FIG. 4 shows the staining profile of four stabletransfectants expressing HA-SSTR2, HA-CXCR4, HA-CCR5 and HA-CCR7,respectively. The mean fluorescence of the four profiles indicates thatthe relative levels of expression were: SSTR2>CXCR4>CCR7>CCR5. CaptureELISA revealed that, as for EL-4 cells, transfected 293 also producedexosomes bearing HA-GPCR (FIG. 5). As illustrated by the resultsobtained with the anti-CD81 antibody, normalized amounts of exosomesproduced by the various transfectants were used. Therefore, the amountsof GPCR detected were directly proportional to the amount of GPCRexpressed per exosome. Remarkably, the same ranking with regard to therelative levels of GPCR expressed at the cell surface and on exosomeswas found, with SSTR2>CXCR4>CCR7>CCR5.

Conclusion: Trans-membrane proteins including GPCR that do not occurnaturally on exosomes can be detected on exosomes following transfectionof cells with membrane protein encoding DNA. This phenomenon does notrequire fusion of the trans-membrane protein sequence withexosome-targeting sequences and appears to be recipient cell-dependent.It may be due to over-expression of recombinant trans-membrane proteinand could serve as an alternative pathway of secretion of excessreceptors. It should be noted that only a fraction of the recombinantprotein produced by transfected cells is expressed on exosomes, the bulkof the proteins being directed toward the cell surface. Regardless, thismethod allows expression of detectable amounts of full-length receptoron exosomes and therefore may be used for exosome display oftrans-membrane proteins that otherwise are not found on exosomes or forthe further enrichment of known exosomal trans-membrane proteins.

EXAMPLE 3 Method for the Generation of Exosomes Expressing RecombinantMHC I/Peptide Complex

The a chain of Human Leukocyte Antigen (HLA) A201 cDNA derived fromA2-positive human leucocytes by reverse transcription was amplified byPCR using the primers HLA2f1 (SEQ ID No. 17) and HLA2r2 (SEQ ID No. 18).The primers were extended at their 5′ end to contain a Hind mrestriction site for HLA2f1 and a BstB I restriction site for HLA2r2.The PCR product was digested with both Hind III and BstB I enzymes andligated into pcDNA6-Myc/His that was precut with Hind III and BstBI I toyield pcDNA6-A2-Myc/His (SEQ ID No. 19).

Similarly, the β chain of HLA (β2-microglobulin or β2M) sequence wasamplified by RT-PCR using the primers β2-MICf1 (SEQ ID No. 20) andβ2-MICr3 (SEQ ID No. 21) and was cloned into pcDNA6-Myc/His to yieldpcDNA6-β2M (SEQ ID No. 22). The β2M insert in pcDNA6-β2M was thensub-cloned into pcDNA3, which contains a different antibiotic resistancegene than pcDNA6. For this purpose, the insert was amplified with theprimers β2-MICf1 and pcDNA6r4 (SEQ ID No. 23), a primer with a Not I 5′end extension. Following Hind III/Not I digestion, the PCR product wasligated into pcDNA3 precut with the same enzymes to yield pcDNA3-β2M.

The human 293 cells were transfected with both pcDNA6-A2-Myc/His andpcDNA3-β2M by electroporation as described above. Two days aftertransfection, cells were placed under double antibiotic selection, i.e.2 μg/ml Blasticidin and 250 μg/ml Neomycin (G418), to establish stabledouble transfectant expressing both A2 and β2M. After 2 weeks underselection, a bulk population of cells was expanded to prepare exosomesas described above. Expression of human A2 and β2M on normalized exosomesamples was assessed by capture-ELISA as described in Example 2. Peptideloading of HLA-A2 on 293-derived exosomes was verified using the DirectLoading method which has been shown to yield functional MHC ClassI/peptide complex (WO01/82958). Briefly, samples were incubated at pH 5with 100 μg/ml biotinylated reference Hepatitis B-peptide FLPSDCFPSV and20 μg/ml β2M (Sigma) for 1 hour. Exosomes were then lysed in 1% NP40 andanalyzed by capture-ELISA using an anti-MHC Class I antibody as captureantibody and Streptavidin-labeled Europium to detect bound biotinylatedreference peptide. Results of capture ELISA measuring recombinantprotein expression and peptide loading are shown FIGS. 6 and 7,respectively.

Results: Transfection of 293 cells with HLA-A2-encoding plasmid resultedin the expression of recombinant A2 in exosomes produced by transfectedcells (FIG. 6). As expected, parental 293-derived exosomes alsocontained endogenous HIA-A2 since 293 is a human HLA-A2⁺ cell line.However, analysis using normalized samples indicates that recombinantexosomes contained ˜5 times more HLA-A2 than the parental exosomes. Incontrast, the amount of β2M was similar in parental and recombinantexosomes, although the overall expression of β2M by recombinant 293cells increased significantly (data not shown). Because the β chain ofMHC Class I complex is a soluble protein, its association with exosomesis mediated through its interaction with the a chain only. Therefore ourresults suggest that the total amount of αβchain complex on exosomesremains constant whereas the subtype of α chain is shifted towardsrecombinant HLA-A2 subtypes. As shown FIG. 7, A2-specific referencepeptide could be detected on recombinant exosomes whereas measurementswere only slightly above background when using parental exosomes.Background level of peptide detection in this assay was determined usingexosomes produced by DC from an A2⁻ donor (A2 negative Dex on FIG. 7).This data indicates that recombinant HLA-A2 can be loaded by A2-specificpeptides using the direct loading method.

Conclusion: Transfection of exosome-producing cells with MHC ClassI-encoding plasmid results in the expression of recombinant MHC Class Imolecules on exosomes. This method allows the preparation of exosomescarrying a unique MHC Class I subtype. Combined with the Direct Loadingmethod, it provides a powerful mean to produce exosomes enriched insubtype-specific MHC Class I peptide complex. These exosomes are potentvehicle for the preparation of antibodies restricted to specific MHCClass I peptide complex.

EXAMPLE 4 Method for the Generation of Enhanced Exosome Displaying CD40LActivity

Human CD40L cDNA was amplified by RT-PCR of activated-T cell RNA usingprimers CD40Lf8 (SEQ ID No. 24) and CD40Lr10 (SEQ ID No. 25). The primerCD40Lf8 was extended at its 5′ end to include a BamH I restriction sitewhereas CD40Lr10 contained a BstB I 5′end extension. The PCR product wasdigested with BamH I and BstB I and ligated into pcDNA6-Myc/His precutwith the same restriction enzymes to yield pcDNA6-CD40L (SEQ ID No. 26).The CD40L insert of pcDNA6-CD40L was then used as template to generate a5′-end and a 3′-end overlapping fragment. The 5′-end fragment wasgenerated using the primer CD40Lf8 and CD40Lr21 (SEQ ID No. 35) whereasthe 3′-end fragment was generated using the primer CD40Lf20 (SEQ ID No.36) and CD40Lr10. CD40Lf20 and CD40Lr21 are complementary to each otherand derived from the sense and anti-sense region of CD40L thatencompasses the sequence encoding residues 112 and 113 of CD40L.Proteolytic cleavage at this site mediates the release of soluble CD40L.The primers were designed to change residues 112 and 113, a Glutamicacid and Methionine in wild type CD40L, into two Glycines to produce amutated form of CD40L that cannot be cleaved at this site and remains inits trans-membrane form. The full-length sequence encoding mutated CD40Lat position 112 and 113 (mutCD40L) was generated by PCR using a mixtureof 5′- and 3′-end fragments as template and the primers pairCD40Lf8/CD40Lr10. The resulting product was cloned into pcDNA6 as aboveto yield pcDNA6-mutCD40L (SEQ ID No. 37). pcDNA6-CD40L andpcDNA6-mutCD40L encode full-length CD40L (SEQ ID No. 34) and mutCD40L(SEQ ID No. 27), respectively. Recombinant exosomes derived fromtransfected 293 cells were prepared as described in Example 2 and 3.Expression of recombinant CD40L and mutated CD40L was monitored byWestern Blot analysis as described in Example 1 and 2 using a rabbitanti-CD40L antibody (Santa Cruz Biotechnology) as detecting antibody. Anassay was also performed to assess whether recombinant 293 acquiredCD40L-like activity by stimulating dendritic cell (DC) maturation.Exosomes were normalized using the cross-capture ELISA described inExample 2 based on 1) the amount of CD81 and 2) the amount of CD40L theyexpressed. Normalized amounts of exosomes were added to 7-day DCcultures grown in IL4/GM-CSF-containing AIM V medium. After overnightculture, DC maturation was evaluated by measuring CD83 expression at thesurface of the cells. This was achieved by standard FACS analysis asdescribed in Example 2 using a monoclonal anti-CD83 antibody conjugatedto FITC. Double staining with anti-CD83 and anti-CD86 antibodies wasperformed to identify the DC population (CD86-positive cells) that alsoexpresses CD83. The results of Western blot analysis for CD40Lexpression and FACS analysis for DC maturation experiments are shown inFIGS. 8 and 9, respectively.

Results: Western blot analysis of recombinant exosomes revealed thattransfection of 293 cells with pcDNA6-CD40L and pcDNA6-mutCD40L resultedin the expression of CD40L expression in exosomes (FIG. 8). Exosomesderived from CD40L-expressing 293 cells contained the full-length CD40L(FL on lane 2) as well as the products of its proteolytic cleavageconsisting of the soluble form of CD40L (SF on lane 2) and the remainingN-terminal extremity that contains the trans-membrane domain of CD40L(TM on lane 2). In contrast, exosomes derived from mutCD40L-expressing293 cells contained only the full-length form (FL on lane 3). No proteinwas detected in exosomes from parental 293 cells (lane 1).

Cell staining and FACS analysis revealed that the number of DC positivesfor CD83, a marker of DC maturation, increased when DC were culturedwith exosomes bearing CD40L and mutCD40L whereas parental exosomes hadno effect (FIG. 9). Moreover, exosomes bearing mutCD40L showed superioractivity whether equal amounts of exosomes or CD40L bound to exosomeswas used in the assay. Indeed, DC maturation was detected when 200 ng/mlmutCD40L bound to exosomes was used in the assay. In contrast, the sameamount of CD40L bound to exosomes or soluble CD40L mixed with parentalexosomes had no detectable effect. Soluble CD40L activity could bedetected in the assay only for concentrations of CD40L of 1 μg/ml andabove.

Conclusion: CD40L, like other trans-membrane proteins (Example 2 and 3)is expressed on exosomes following over-expression of transfected cellswithout requiring fusion to exosome-targeting domains. Recombinantexosomes expressing CD40L also display CD40L activity. Therefore, thepresent invention may be used to enhance exosome immuno-competency. Amutation in the proteolytic cleavage site of CD40L resulted in thefurther increase of exosome immuno-competency. A possible explanationfor this property is that the wild type CD40L on exosomes consists of amixture of full-length and soluble forms of CD40L as well as residualtrans-membrane domain of CD40L, which may hamper the efficaciousformation of the functional trimeric form of CD40L. In this context,preventing the cleavage of CD40L into its fragments results in theproduction of enhanced exosomes displaying higher specific activity.This may be required for vaccine and other research applications usingrecombinant exosomes since circulating proteases might cleave CD40L andinactivate exosomes shortly after injection.

EXAMPLE 5 Method for the Generation of Traceable Exosomes Using GFP/C1C2Chimeric Protein

The cDNA encoding the fluorescent protein GFP minus the initiation codonwas cloned between the leader sequence and the C1/C2 domain ofLactadherin to produce LS-GFP-C1/C2 (SEQ ID No. 40). GFP cDNA wasamplified by PCR using pEGFP (Clontech, CA) as template and primersGFPf1 (SEQ ID No. 41) and GFPr2 (SEQ ID No. 42). The primer GFPf1 wasextended at its 5′end to include an EcoR I restriction site whereasGFPr2 contained a Not I 5′end extension. The PCR product was digestedwith EcoR V and Not I and ligated to C1/C2 PCR product precut with Not Iand BstB I. C1/C2 cDNA fragment was generated by PCR usingpcDNA6-hLactlf/His as template and the primers LIDNf40 (SEQ ID No. 43)and LTDNr35 (SEQ ID No. 44). The ligation mix was then added topcDNA6-hLact28-Myc/His (SEQ ID No. 45) precut with EcoR V and BstB I.pcDNA6-hLact28-Myc/His encodes the N-terminus of human Lactadherin thatincludes its leader sequence and EGF domain. It was prepared by ligatingthe product of a PCR using pcDNA6-hLactlf/His as template and theprimers LTDNf15 and LTDNr28 (SEQ ID No. 46). DigestingpcDNA6-hLact28-Myc/His with EcoRV and BstB I released the DNA fragmentbetween the Lactadherin leader sequence and the Myc/His tags which wasreplaced by GFP-C1/C2 DNA to produce pcDNA6-LS-GFP-C1/C2-Myc/His (SEQ IDNo. 39). The control plasmid pcDNA6-LS-GFP-Myc/His (SEQ ID No. 28)encoding LS-GFP-Myc/His (SEQ ID No. 29) was also prepared using asimilar approach where GFPr2 was modified to contain a BstB I extensionand GFP sequence was inserted into precut pcDNA6-hLact28-Myc/Hisomitting the pre-ligation step with C1/C2 PCR product. Clones derivedfrom stable cell lines transfected with the GFP plasmids were prepared,and exosomes were purified as described above for other constructions(Example 1 to 4). Exosomes were assessed for the presence of GFP on thebasis of their fluorescent emission. Measurement was performed onexosomes placed in the wells of a 96-well plate and by fluorescencereading using a Wallac 1420 Victor² plate reader. The results of thismeasurement are shown FIG. 10.

Results: Fluorescence was detected with exosomes derived from cellstransfected with pcDNA6-LS-GFP-C1/C2-Myc/His whereas background levelswere read with exosomes produced by pcDNA6-LS-GFP-Myc/His-transfectedcells (FIG. 10). Western blot analysis revealed that, as expected, thelatter produced recombinant GFP that was released in the culturesupernatant as a soluble protein (data not shown).

Conclusion: These data further confirm that fusion of sequences encodingsoluble proteins to exosome-targeting domains direct expression ofproteins to exosomes. Expression of GFP associated with exosomes can beused to prepare traceable tools for various applications including thesorting of antigen-specific antibody producing cells (see Example 7,below).

EXAMPLE 6 Method for the Generation of Traceable Exosomes by MetabolicLabeling Using Fluorescent Lipids

In this example, traceable exosomes were prepared using fluorescentlipids that metabolically label exosomes. The lipid1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(Lissamine Rhodamine BSulfonyl) (Rh-DOPE, Avanti Polar Lipids) was prepared by diluting anethanol solution of the lipid into sterile PBS to give a finalconcentration of 40 μM. Incremental amounts of the 40 μM stock solutionwere then added to 293 cells cultures to yield 3.0 μM of the fluorescentlipid. Exosomes from day-7 culture we're harvested and purified asdescribed in Example 14. Fluorescence associated with purified exosomeswas assessed by capture-ELISA where samples were added to the well of a96-well plate pre-coated with an anti-CD81 antibody. Following extensivewashing, fluorescence emission at 600 nm upon excitation of the wells at560 nm was read using a Wallac 1420 Victor² plate reader. Controlsamples for this experiment consisted of exosomes derived fromfluorescent lipid-free media (Exo), exosomes derived from fluorescentlipid-free media and incubated with fluorescent lipid-containing mediafor 24 hours (Exo+Rh), fluorescent lipid-containing media alone (Rh) andPBS. Results are shown in FIG. 11.

Results: The production of exosomes by cells exposed to the Rh-DOPElipid (Rh/Exo on FIG. 11) yielded highly fluorescent exosomes. Incontrast, low to background levels of fluorescence were detected whenexosomes were incubated directly with fluorescent lipids (Exo+Rh on FIG.11) or when exosomes were produced in the absence of fluorescent lipid(Exo on FIG. 11). The fluorescence measured was directly associated toexosomes since only background fluorescence was detected in the assaywhen Rh-DOPE-containing media alone was added to the wells (Rh on FIG.11). Production of exosomes by cells exposed to other florescent lipidssuch as1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(Carboxyfluorescein)(Fl-DOPE) also yielded highly fluorescent exosomes (data not shown).

Conclusion: The method described here results in the production oftraceable exosomes. The intensity of fluorescence associated with theseexosomes is far superior to that of exosomes directly labeled withflorescent lipids. Their usage should allow read out in many researchand screening applications with increased sensitivity. Traceableexosomes containing fluorescent lipids are, for instances, a verypowerful tool to select and isolate antigen-specific antibody-producingcells (see Example 7 below).

EXAMPLE 7 Method for Identifying and Isolating Antigen-Specific B Cell

Popliteal Lymph Nodes, cells (PLNC) from mice immunized with recombinantGPCR-expressing exosomes were harvested after four successive injectionsof inoculum in the footpad at 1-week intervals. The inoculum consistedof purified exosomes derived from mouse cells transfected withGPCR-encoding plasmids and prepared as described in Example 2. PLNC werewashed twice in PBS/5% FBS at 4° C. then two aliquots of 10E6 PLNC wereresuspended into 500 μl PBS/5% FBS. Normalized amounts of GFP-expressingexosomes, GFP- and GPCR-expressing exosomes and parental exosomes wereprepared as described in Example 2 and 5. An equal amount of traceablecontrol exosomes (GFP-293) and traceable test exosomes (GFP/GPCR-293)was added to the first and second PLNC sample, respectively. A 10×excess of parental exosomes was added to both samples to blocknon-specific interaction of exosomes with PLNC. An anti-CD19 antibodyconjugated to phycoerythrin was also added to the samples tospecifically select the B cell sub-population in PLNC. The samples wereadjusted to 1 ml final with PBS/5% FBS and incubated for 1 hour at 4° C.Cells were then analyzed by FACS to identify double positives for GFPand CD19. These double positive cells are expected to be antibodyproducing cells, i.e. B cells, and to express antibodies at theirsurface that react specifically with the recombinant GPCR onGFP-exosomes. The results of an experiment performed using CCR7 as modelare shown FIG. 12.

Results: The percentage of GFP-positive cells in the PLNC populationderived from immunized cells was significantly higher when cells wereincubated with GFP/CCR7-293 exosomes than with control GFP-293 exosomes(FIG. 12, Panel A, 0.13% positive cells vs Panel B 0.36% positivecells). Although this percentage is low, a similar trend was observedwhen using PLNC from mice immunized with exosomes expressing other GPCRsuch as CXCR4, CCR5 and SSTR2 and the matching traceable recombinantexosomes (data not shown).

Conclusion: Traceable exosomes represent a convenient tool to identifyantigen-specific antibody-producing cells. Combined with FACS analysisand sorting, they should allow isolating individual cells with definedantigen specificities even at very low frequency of responding cells.

EXAMPLE 8 Subtractive Method for Identifying and Isolating B CellsProducing Epitope-Specific Antibody

In this example, the sorting of antibody-producing cells is extended toepitope-specific antibody-producing cells. The method is similar to thatdescribed in Example 7 with the exception that PLNC of immunized miceare now incubated with an excess of antigen-expressing exosomes insteadof parental exosomes to block B cells producing antibodies reacting withundesirable epitopes on the antigen. In such subtractive sorting, Bcells reacting to neo-epitopes found on mutated antigens orconformational variants of the antigen are free to react with traceableGFP-293 expressing the antigen variant. This approach is bestillustrated for the preparation of restricted antibodies that react withMHC/peptide complex. The preparation of exosomes expressingHLA-A2/peptide complex has been described in Example 3. In order toenrich the PLNC in epitope-specific antibody-producing cells, mice wereimmunized with recombinant exosomes using countralateral injections.Here, the right footpad of mice received six inoculums of mouse-derivedexosomes expressing recombinant A2 at day −3, 0, 3, 10, 17 and 24. Theleft footpad received five inoculums at day 0, 3, 10, 17 and 24 of thesame exosomes loaded with a MART1-derived peptide (SEQ ID No. 30). Rightand left PLNC were prepared separately for staining as described inExample 7. An equal amount of traceable control exosomes (GFP/A2-293) ortraceable test exosomes (GFP/A2/MART1-293) was added to both PLNCpopulations, respectively. A 10× excess of A2-293 was added to allsamples to mask B cells producing antibody reacting with A2 alone. Theresults of a FACS analysis with these samples are shown FIG. 13.

Results: The percentage of GFP-positive cells in the left PLNCpopulation derived from the side of the mice immunized withA2/MART1-exosomes was significantly higher when cells were incubatedwith GFP/A2/MART1-293 exosomes than with GFP/A2-293 exosomes (FIG. 13,Panel A, 0.83% positive cells vs Panel B, 0.33% positive cells). Incontrast, no significant difference could be detected when comparing theright PLNC incubated with GFP/A2/MART1-293 and GFP/A2-293 (FIG. 13,Panel C, 0.49% positive cells vs Panel D, 0.39% positive cells).

Conclusion: The subtractive method that uses traceable exosomesdescribed here is a powerful approach to identify epitope-specificantibody-producing cells. Combined with FACS analysis and sorting, itenables isolation of individual cells with defined epitopespecificities. Subtractive sorting is amenable, for instances, toisolating B cells producing restricted antibodies that react withMHC/peptide complex.

EXAMPLE 9 Method for Retrieving Immunoglobulin Sequences by RT-PCR

Cells producing antibodies with defined antigen or epitope specificitiesand identified by FACS as described in Example 7 and 8, were sortedindividually in the wells of a 96-well plate containing 25 μl ofice-cold lysis buffer (Cells to cDNA II kit, Ambion). RNases wereheat-inactivated by incubating the 96-well plate at 75° C. for 15minutes. Samples were then treated with 1 Unit of DNase I to eliminategenomic DNA. The resulting samples contained RNA that was transferredinto a new plate for cDNA synthesis using Reverse Transcriptase andoligo-dT (SuperScript II, Invitrogen). cDNA encoding the variable regionof the light and heavy chains of immunoglobulins that contains thecomplementary determinant regions involved in contacting antigens wasamplified by nested-PCR. A mixture of forward primers including IGLKf1(SEQ ID No. 31), IGLKf2 (SEQ ID No. 32), IGLKf3 (SEQ ID No. 33), IGLKf4(SEQ ID No. 34), IGLKf5 (SEQ ID No. 35) and the reverse primer IGKr15(SEQ ID No. 36) were used for a first round PCR of light chainsequences. A second round PCR (nested-PCR) was performed using analiquot of the first round PCR as template and the degenerated primersIGKf6 (SEQ ID No. 37) and IGKr15. Similarly, a mixture of forward primerincluding IGLHf1 (SEQ D No. 38), IGLHf2 (SEQ ID No. 39) and IGLHf3 (SEQID No. 40) and the reverse primer IGHr5 (SEQ ID No. 41) were used for afirst round PCR of heavy chain sequences. A second round PCR(nested-PCR) was performed using an aliquot of the first round PCR astemplate and the degenerated primers IGHf11 (SEQ ID No. 42) and IGHr13(SEQ ID No. 43). The results of amplification of light chain sequencesusing cDNA from cells sorted individually based on their ability to bindfluorescent exosomes in an antigen-specific manner is shown FIG. 14.

Results: Two rounds of PCR yielded detectable amounts of PCR productsderived from several single cell cDNA (FIG. 14, lane 2, 5, 6, 8 and 13).Sequencing of these PCR products confirmed that they encode the variableregions of immunoglobulins. Several possibilities may explain the lackof PCR products in the empty lanes of FIG. 14. One possibility is thatthe PCR cycling conditions and primer pairs used here were not optimumto amplify efficiently the variable sequences of immunoglobulinexpressed by the single cell of the original well. Indeed, everyvariable region is unique and may require different PCR conditions foramplification. Other primer combinations and cycling conditions may betried with each cDNA for optimum results. Another possibility is thatthe cell sorted in the original well was a non-B cell or a resting Bcell. These cells which can be sorted due to non-specific interactionsof traceable exosomes with PLNC (see background staining in Example 7and 8) contain no or too low levels of RNA encoding immunoglobulins fordetection by RT-PCR. This emphasizes that the sensitivity of the methodproposed here to retrieve variable sequences of immunoglobulin may avoidthe further carry over of cDNA from non-antibody-producing cells.

Conclusion: Variable sequences of immunoglobulin from single cellsisolated based on their ability to interact with traceable exosomes inan antigen-specific manner can successfully be retrieved by classicalRT-PCR. The PCR products can then be cloned into expression vectors asdescribed in Example 10, below.

EXAMPLE 10 Method for Cloning of Variable Sequences of Immunoglobulinsinto Expression Vectors

Expression vectors were constructed with embedded portions of heavy andlight chain antibody sequences to receive the variable region PCRproducts generated in Example 9. Cloning of variable region PCR productsin these vectors yields full-length heavy and light chain sequences.Unlike classical methods of insert cloning, the cloning strategy usedhere does not introduce mutations at the cloning sites. This method isadvantageous since mutations at either extremity of the variable regionof immunoglobulins may modify antigen affinity and specificity. It usestype “IIs” restriction enzymes such as BfuAI, BsaI and BsmBI that cutoutside the sequence recognition site, leaving the recognition sequenceon one side of the cut. A cassette containing two such restriction sitesallows the release of inserts framed by the two sites, leaving only theembedded antibody sequence. Inserts for cloning are generated to includesimilar sites at both ends with sequence extremities compatible to theembedded antibody sequence. Thereby, ligation of inserts occurs viaannealing of gene-specific sequences without the need to insert ormodify bases matching the recognition sites of restriction enzymes.

Separate vectors were constructed for antibody heavy and light chains.These contained the leader sequence and constant regions of aprototypical heavy and light chain, respectively, flanking a linker-freecloning cassette. Mouse spleen cDNA encoding light chain sequences wasamplified to generate an upstream leader sequence fragment using primersIGKf1 (SEQ ID No. 44) and IGKr2 (SEQ ID No. 45) and a downstreamconstant region fragment using primers IGKf12 (SEQ ID No. 46) and IGKr4(SEQ ID No. 47). IGKr2 and IGKf12 were designed to contain overlappingsequences encompassing the linker-free cloning cassette. The completeDNA fragment including leader sequence, linker-free cloning cassette andconstant region was generated by a second PCR using the upstream anddownstream fragments as template and primers IGKf1 and IGKr4. Theseprimers contained 5′ end extensions with a Hind III restriction site forIGKf1 and a BstB I for IGKr4. Following digestion with Hind III and BstBI, the PCR product was cloned into pcDNA6-Myc/His precut with the sameenzymes yielding pcDNA6-LCJ-Myc/His (SEQ ID No. 48). pcDNA6-HCJ-Myc/His(SEQ ID No. 49) encoding the heavy chain equivalent ofpcDNA6-LCJ-Myc/His was prepared using the same approach. Here, the firstround of PCR was performed with primer pairs IGHf1(SEQ ID No.50)/IGHr2(SEQ ID No. 51) and IGHf15(SEQ ID No. 52)/IGHr4(SEQ ID No. 53).The second round PCR was performed with IGHf1 and IGHr4.

Another construction was prepared to express the heavy and light chainsof antibody in a single vector. This is to ensure that any transfectedcell produces both the heavy and light chains. For this purpose, aninternal nbosome entry site (IRES) and a second multiple cloning site(mcs2) derived from pIRES (Clontech) was cloned downstream of LCJ inpcDNA6-LCJ-Myc/His. The IRES/mcs2 insert was generated by PCR usingpIRES as template and primer pIRf1(SEQ ID No. 54) and pIRr2 (SEQ ID No.55). After digestion with BstBI and Age I, the PCR product was ligatedinto pcDNA6-LCJ-Myc/His precut with the same enzymes, yieldingpcDNA6-LCJ-IRES. The cloning of heavy and light chain variable regionsinto pcDNA6-LCJ-IRES was then performed in two steps. First, the lightand heavy chains was cloned into pcDNA6-LCJ-IRES and pcDNA6-HCJ-Myc/His,respectively. Second, the full-length heavy chain prepared in the firststep in pcDNA6-HCJ-Myc/His was sub-cloned into pcDNA6-L-IRES alsoprepared in the first step and already encoding the full-length lightchain.

An experiment was performed to verify that the PCR products generated inExample 9 and cloned into expression vectors using the method describedhere yielded immunoglobulins. The PCR products HV-M90/12 (SEQ ID No. 56)and LV-M90/12 (SEQ ID No. 57) encoding the variable regions of heavy andlight chains sequences, respectively, derived from a single cellisolated as described in Example 8 were used as model. BsmB Irestriction sites were introduced at the extremities of each DNAfragment by a 10-cycle PCR using primers IGKf6 and a 1:1 mix ofIGKr10(SEQ ID No. 58):IGKr11 (SEQ ID No. 59) for LV-M90112 and primersIGHf11 and a 1:1:1 mix of IGHr16(SEQ ID No. 60)/IGHr17(SEQ ID No.61)/IGHr18(SEQ ID No. 62) for HV-M90/12. These primers were designed toleave overhang extremities complementary to the junctions of theembedded antibody sequences in the recipient expression vectors.Following digestion with BsmB I, LV-M90/12 was cloned intopcDNA6-LCJ-IRES and pcDNA6-LCJ-Myc/His precut with the same enzymeyielding pcDNA6-L-M90/12-IRES (SEQ ID No. 63) andpcDNA6-L-M90/12-Myc/His (SEQ ID No. 64), respectively. HV-M90/12 wasfirst cloned into pcDNA6-HCJ-Myc/His precut with BsmB I yieldingpcDNA6-H-M90/12-Myc/His (SEQ ID No. 65). The full-length H-M90/12 heavychain sequence was then sub-cloned into mcs2 of pcDNA6-L-M90/12-IRES. Todo so, Xba I and Not I restriction sites were introduced by PCR at the5′ and 3′ end, respectively, of H-M90/12 using pcDNA6-H-M90/12-Myc/Hisas template and the primers pIRHf1 (SEQ ID No. 66) and pIRHr2 (SEQ IDNo. 67). Following digestion with Xba I and Not I, H-M90/12 was clonedinto pcDNA6-L-M90/12-IRES precut with the same enzymes yieldingpcDNA6-L+H-M90/12. CHO cells were transfected with either empty plasmid,pcDNA6-L+H-M90/12 or a 1:1 mix of pcDNA6-L-M90/12-Myc/His:pcDNA6-H-M90/12-Myc/His. Day-3 culture supernatants were harvested andtested for immunoglobulin production by capture-ELISA as described inExample 2 using a donkey anti mouse IgG antibody (JacksonImmunoResearch) as capture antibody and a rabbit anti-mouse IgG asdetecting antibody. A standard curve was established using incrementalamounts of purified mouse IgG. The results of this ELISA are shown FIG.15.

Results: Recombinant immunoglobulins were successfully detected in thesupernatant of CHO-transfected cells (FIG. 15). Moreover, transfectionwith expression vector encoding both light and heavy chains ofantibodies was superior to co-transfection with plasmids encoding eachchain separately.

Conclusion: The cloning strategy for the construction of plasmidsencoding full-length immunoglobulin described here yields expressionvectors producing significant amounts of recombinant immunoglobulinswithout introducing insertion or mutations in the variable regions ofthe antibody produced. Overall, Example 7 to 10 support that antibodysequences derived from individual cells producing antibodies withdefined specificity can be retrieved, cloned and expressed asrecombinant proteins using the methods described in this invention. Theantigen specificity of the recombinant antibody produced can then beevaluated in binding assays, preferably using recombinant exosomes assource of antigen.

EXAMPLE 11 Isolation of Antibody-Secreting Hybridoma Using BiotinylatedExosomes

Spleen cells of mice immunized as described in Example 8 were fused withthe mouse myeloma cell line Sp2/0 using standard method of hybridomapreparation. Following fusion, cells were grown as a bulk-culture inmedia supplemented with Azaserine for selection of hybridoma. At day-8of the culture, cells were harvested and incubated successively withbiotinylated anti-CD45 antibody and Streptavidin. Cells were thenseparated into two equal fractions, i.e. antibody-trap positive(AbTrap+) and negative (AbTrap−) fractions. The antibody trap of theAbTrap+ fraction was completed by incubating cells with biotinylatedanti-mouse IgG antibody. Both cell fractions were then incubatedovernight in culture media at 37° C. under a 10% CO₂ atmosphere to allowhybridoma antibody secretion and capture by the antibody trap. Hybridomasecreting antibodies that were trapped at their cell surface anddisplayed desired antigen specificities were isolated by FACS followingincubation with biotinylated exosomes. These traceable exosomes wereprepared by incubating exosomes with chemically reactive biotin bearingtetrafluorophenol (TFP) conjugated to biotin via a polyethylene oxide(PEO) linker. TFP activated biotin reacts with primary amines onexosomes to form a covalent linkage. Hybridroma bearing biotinylatedexosomes at their surfaces were detected using Streptavidin conjugatedto the fluorophore Alexa488. FACS analysis of AbTrap + and − hybridomaproducing anti-exosome antibodies is shown FIG. 16.

Results: Incubation of biotinylated-exosomes with AbTrap− cells resultedin background fluorescence associated with these cells (FIG. 16, panel1). In contrast, ˜25% of these cells were positive when bearing the fullAbTrap (FIG. 16, panel 2).

Conclusion: The antibody trap designed here successfully capturedantibodies secreted by hybridoma, which enable hybridoma isolation byFACS. In addition, these results show that biotinylated exosomes aresuitable traceable exosomes to isolate antibody-producing cells in anantigen-specific manner.

REFERENCES

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1. A method of isolating single antibody-producing particles havingspecificity for a selected antigen, comprising: 1) preparing exosomesdisplaying a selected antigen and a marker, said antigen being fused toan exosome targeting polypeptide, and said exosome targeting polypeptidebeing a partial murine lactadherin sequence selected from the groupconsisting of amino acid residues 111-266, 109-266, 271-426, 111-426 and109-426 of SEQ ID NO: 1; 2) contacting said exosomes of step 1 with anantibody-producing particles repertoire; and, 3) identifying andisolating single antibody-producing particles reacting with saidexosomes.
 2. The method of claim 1, wherein said antibody-producingparticles are antibody-producing cells and wherein saidantibody-producing particles repertoire is a repertoire ofantibody-producing cells.
 3. The method according to claim 2, whereinsaid antibody-producing cells are selected from the group consisting ofplasma cells, hybridoma and lymphocytes.
 4. The method according toclaim 1, wherein said method comprises: 1) providing antibody-producingcells; 2) preparing exosomes displaying said antigen and a marker; 3)suspending the antibody-producing cells of step 1 with the exosomes ofstep 2; and, 4) identifying and isolating single antibody-producingcells reacting with said exosomes.
 5. The method according to claim 4,wherein said antibody-producing cells are lymphocytes collected fromnon-human animals immunized with said antigen.
 6. The method accordingto claim 1, wherein said method comprises: 1) providingantibody-producing cells; 2) preparing the exosomes displaying saidantigen and said marker; 3) suspending the antibody-producing cells ofstep 1 with the exosomes of step 2; and, 4) identifying and isolatingsingle antibody-producing cells reacting with said exosomes; and whereinsaid antibody-producing cells are antibody-secreting cells and themethod further comprises, before the step of suspending theantibody-secreting cells with the exosomes, the step of incubatingantibody-producing cells with a first biotinylated-antibody against aubiquitous cell surface marker, streptavidin and a second biotinylatedantibody directed against immunoglobulin of said antibody-producingcells.
 7. A method of isolating single antibody-producing cells havingspecificity for a selected antigen, comprising: 1) preparing immunogenicexosomes displaying at least one antigen or an epitope thereof, saidantigen or said epitope being fused to an exosome targeting polypeptide,and said exosome targeting polypeptide being a partial murinelactadherin sequence selected from the group consisting of amino acidresidues 111-266, 109-266, 271-426, 111-426 and 109-426 of SEQ ID NO: 1;2) raising an antibody response by immunizing a non-human animal withsaid immunogenic exosomes; 3) collecting lymphocytes from said immunizedanimal; 4) preparing exosomes displaying the said antigen or an epitopethereof of step 1 and a marker; 5) suspending the lymphocytes of step 3with the exosomes of step 4; and, 6) identifying and isolating singleantibody-producing cells reacting with the exosomes of step
 4. 8. Themethod of claim 1, wherein said antibody-producing particles areantibody-producing phages or yeasts and wherein said antibody-producingparticles repertoire is a repertoire of antibody-producing phages oryeasts.
 9. The method according to claim 1 wherein said method furthercomprises the following steps: a) recovering DNA or RNA from saidselected antibody producing particles, b) amplifying the nucleic acidsequence encoding immunoglobulin sequences or portions thereof, c)cloning the amplified nucleic acid sequence into an expression vector toproduce proteins with desired antigen specificity.
 10. The methodaccording to claim 7, wherein said immunogenic exosomes further displayimmune accessory molecules.
 11. The method according to claim 10,wherein said immune accessory molecule is an adjuvant polypeptide. 12.The method according to claim 10, wherein said immune accessorymolecules are fused or cross-linked to an exosome targeting polypeptide.13. The method according to claim 10, wherein said immune accessorymolecules have at least one transmembrane domain and are incorporatedinto immunogenic exosomes by over expression into the exosome-producingcells.
 14. A method of isolating particles producing a single antibodyspecific for a variant antigen from an antibody-producing particlesrepertoire comprising: 1) preparing a first population of exosomesdisplaying said variant antigen and a marker, said variant antigen beingfused to an exosome targeting polypeptide, and said exosome targetingpolypeptide being a partial murine lactadherin sequence selected fromthe group consisting of amino acid residues 111-266, 109-266, 271-426,111-426 and 109-426 of SEQ ID NO: 1; 2) preparing a second population ofexosomes displaying the wild-type antigen and not displaying saidmarker, said wild-type antigen being fused to an exosome targetingpolypeptide, and said exosome targeting polypeptide being a partialmurine lactadherin sequence selected from the group consisting of aminoacid residues 111-266, 109-266, 271-426, 111-426 and 109-426 of SEQ IDNO: 1; 3) suspending said antibody-producing particles repertoire withthe first and second populations of exosomes, the second populationbeing in excess; and, 4) identifying and isolating singleantibody-producing particles reacting with the exosomes of step
 1. 15.The method according to claim 14, wherein said method comprises: 1)collecting lymphocytes from a non-human animal immunized with saidvariant antigen; 2) preparing exosomes displaying said variant antigenused for the animal immunization and said marker; 3) preparing exosomesdisplaying the wild-type antigen and not displaying said marker; 4)suspending the lymphocytes of step 1 with the exosomes displaying saidvariant antigen and marker of step 2 and with an excess of the exosomesdisplaying said wild-type antigen of step 3; and, 5) identifying andisolating single antibody-producing cells reacting with the exosomes ofstep
 2. 16. A method of isolating cells producing a single antibodyspecific for a variant antigen from an antibody-producing particlesrepertoire comprising: 1) preparing immunogenic exosomes displaying avariant antigen, said variant antigen being fused to an exosometargeting polypeptide, and said exosome targeting polypeptide being apartial murine lactadherin sequence selected from the group consistingof amino acid residues 111-266, 109-266, 271-426, 111-426 and 109-426 ofSEQ ID NO: 1; 2) raising an antibody response by immunizing a non-humananimal with the said immunogenic exosomes; 3) collecting lymphocytesfrom said immunized animal; 4) preparing exosomes displaying saidvariant antigen of step 1 and a marker; 5) preparing exosomes displayingthe wild-type antigen and not displaying said marker; 6) suspending thelymphocytes of step 3 with the exosomes displaying said variant antigenand marker of step 4 and with an excess of the exosomes displaying saidwild-type antigen of step 5; and, 7) identifying and isolating cellsproducing a single antibody specific for a variant antigen reacting withthe exosomes of step
 4. 17. The method according to claim 14, whereinsaid variant antigen is a mutated antigen.
 18. The method according toclaim 14, wherein said variant and wild-type antigens are differentconformational states of any protein, including an enzyme.
 19. Themethod according to claim 1, wherein said marker is a detectablemolecule selected from tags, biotin, enzyme, and fluorescent molecules.20. The method according to claim 19, wherein said marker is fused orcross-linked to an exosome targeting polypeptide.
 21. The methodaccording to claim 19, wherein said marker has a transmembrane domainincorporated into immunogenic exosomes by over expression intoexosome-producing cells.
 22. The method according to claim 19, whereinsaid marker is a labeled lipid.
 23. The method according to claim 22,wherein said labeled lipids are fluorophore-conjugated lipids.
 24. Themethod according to claim 1, wherein said antigen is a receptor.
 25. Themethod according to claim 24, wherein said receptor is a GPCR (GProtein-Coupled Receptor).
 26. The method according to claim 1, whereinsaid antigen is any protein or compounds other than polypeptides. 27.The method according to claim 11, wherein said adjuvant polypeptide is acytokine.
 28. The method of claim 27 wherein said cytokine is selectedfrom the group consisting of GM-CSF, IL-2 and CD40L.
 29. The method ofclaim 27 wherein cytokine is a mutated CD40L.
 30. The method of claim 29wherein said mutated CD40L contains a mutation which prevents cleavageand release of soluble CD40L.
 31. The method according to claim 22,wherein said labeled lipid is incorporated in an exosome.
 32. The methodaccording to claim 26, wherein said protein is a receptor or an enzyme,and said compounds other than polypeptides are glycolipids,polysaccharides, drugs or organic chemicals.